Nuclear thyroid hormone receptor-interacting polypeptides and related molecules and methods

ABSTRACT

Disclosed is a method for determining whether a test protein is capable of interacting with a nuclear hormone receptor protein. The method involves: (a) providing a host cell which contains (i) a reporter gene operably linked to a protein binding site; (ii) a first fusion gene which expresses a first fusion protein, the first fusion protein including a nuclear hormone receptor protein covalently bonded to a binding moiety which is capable of specifically binding to the protein binding site; and (iii) a second fusion gene which expresses a second fusion protein, the second fusion protein including the test protein covalently bonded to a weak gene activating moiety; and (b) determining whether the test protein increases expression of the reporter gene as an indication of its ability to interact with the nuclear hormone receptor protein. Such an interaction may be hormone dependent, hormone independent, or hormone sensitive. Also disclosed is purified DNA encoding thyroid hormone receptor-interacting proteins and the polypeptides expressed from such DNA.

This is a divisional of application Ser. No. 08/222,719, filed Apr. 4,1994, which is a continuation-in-part of application Ser. No. 07/969,136filed on Oct. 30, 1992 now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to receptor proteins.

The diverse physiological and developmental effects of thyroid hormonereceptor (for example, T3) are mediated by the three hormone-bindingisoforms of the T3 receptor: TRα1, TRβ1, and TRβ2. The effects of thehormone are the consequences of changes in expression of a wide range oftarget genes that result from T3 binding to these receptors. While it isunknown how binding of the ligand to the receptor actually causes suchchanges in gene expression, the basic effects on the rate oftranscription are believed to be a consequence of direct or indirectprotein-protein contacts between the TRs and components of basictranscriptional apparatus, such as RNA polymerase or associatedproteins. In addition, interactions of TRs with other transcriptionfactors are thought to result in a variety of complex combinatorialregulatory effects.

In recent years there has been very rapid progress in unravelling themost basic aspects of the mechanism of T3 action in the control of geneexpression (see Brent et al., Ann. Rev. Physiol. 53:17-35, 1991 forrecent review). It is now clear that the T3 receptors are transcriptionfactors that belong to a related superfamily of nuclear hormonereceptors. This family of proteins interacts not only with diverseligands but also with a complex array of similar DNA binding sites. Likeother DNA binding transcription factors, the TRs function by increasing(or, in some cases, decreasing) the rate of transcription initiationfrom a linked promoter.

Other details of the mechanisms that cause such alterations remainunclear and are the focus of intense study in a number of systems (seeLewin, Cell 61:1161-1164, 1990; Ptashne, Sci. Am. 260:40-47, 1989;Ptashne, and Gann, Nature 346:329-331, 1990, for reviews). However, twobroad themes are evident. The first is that transcription factors ingeneral are frequently modular, composed of distinct domains withseparate DNA binding and transcriptional regulatory functions. With TRs,for example, it is apparent that the DNA binding and ligand bindingdomains are quite separate, and experiments with chimeric receptors makeit clear that the T3 dependent activation of gene expression can betransferred to heterologous DNA binding domains (see, e.g., Holloway,Proc. Natl. Acad. Sci. U.S.A. 87:8160-8164, 1990; Thompson and Evans,Proc. Natl. Acad. Sci. U.S.A. 86:3494-3498, 1989).

A second theme is that the functions of transcription factors arebelieved to be a consequence of protein-protein interactions with thebasic transcriptional apparatus. It is thought that these interactionsare mediated by proteins called coactivators or adaptors (see Ptashneand Gann, Nature 346:329-331, 1990). These poorly characterized proteinsact as bridges between the transcriptional activation domain that istethered to the DNA by the transcription factor and the RNA polymerasecomplex bound at the initiation site. Via unknown mechanisms, thisinteraction leads to an increase in promoter activity.

Protein-protein contacts are also essential for a surprisingly diversearray of positive and negative interactions between transcriptionfactors. Recent results in several systems indicate that this mechanismleads to complex regulatory networks that allow cross talk betweenvarious signalling pathways. In the case of TRs, three such interactionshave been described to date. The first is the heterodimeric interactionof TRs with the related RXRs (Bugge et al., EMBO J 11:1409-1418, 1992;Kliewer et al., Nature 355:446-449, 1992; Lied et al., Cell 68:377-395,1992; Marks et al., EMBO J 11:1419-1435, 1992; Yu et al., Cell67:1251-1266, 1991; Zhang et al., Nature 355:441-446, 1992). TR/RXRheterodimers show higher DNA binding affinity to thyroid hormoneresponse elements (i.e., T3RE sites) initially characterized as bindingTR homodimers (see, e.g., Williams et al., J. Biol. Chem.266:19636-19644, 1991), but heterodimerization does not appear to altersite specificity.

A second, less direct interaction is reflected in the mutuallyantagonistic effects of the TRs and the c-jun and c-fos protooncogenes(Desbois et al., Cell 67:731-740, 1991; Zhang et al., Mol. Cell. Biol.11:6016-6025, 1991). The heterodimeric complex of these two leucinezipper transcription factors is frequently referred to as AP-1, althoughthe jun-jun homodimers and other complexes containing related but lesswell characterized proteins can also bind the consensus AP-1 site. Suchsites are also referred to as TPA response elements (i.e., TREs) (heredistinguished from T3REs) because the induction of protein kinase Cactivity by TPA or other phorbol esters results in a very rapidinduction of AP-1 activity (reviewed in (Curran and Franza, Cell55:395-397, 1988). The activity of the TRs is antagonized bycoexpression of active jun or fos, and the TRs exert a complimentaryinhibition of jun and fos activity. Although the mechanism of thisinteraction is unknown, it does not require the presence of overlappingDNA binding sites. Thus, TRs can antagonize TPA response on a promoterthat does not contain a T3RE, and jun and fos can antagonize T3 responseon a promoter that does not include a TRE. Interestingly, although TRsare always nuclear and are able to bind T3REs whether or not hormone ispresent, the antagonistic function is only observed when T3 is present.

The antagonistic interaction with jun and fos is also observed withother members of the superfamily, including RARs (Desbois et al., Cell67:731-740, 1991; Schule et al., Proc. Natl. Acad. Sci. U.S.A.88:6092-6096, 1991) and GRs (Jonat et al., Cell 62:1189-1204; Schule etal., Cell 62:1217-1226, 1990; Yang-Yen et al., Cell 62:1205-1215, 1990).The GR interaction was the first described and has been the bestcharacterized, but the biochemical basis for the effect remainsuncertain (see Ponta et al., Acta 1129:255-261, 1992 for a review).Despite the potential importance of this apparent cross-talk betweennuclear hormone receptors and the protein kinase C signalling pathway,its physiologic impact also remains unclear.

Finally, TRs have also been reported to interact both functionally andbiochemically with the cell-type specific transcriptional activatorPit-1 (Schaufele et al., Mol. Endocrinol. 6:656-665, 1992). In contrastto the antagonistic effects of TRs and AP-1, this interaction apparentlyleads to synergistic activation.

These distinct mechanisms for the modulation of transcriptionalactivation remain quite unclear. It is apparent that the identificationand characterization of proteins capable of interacting specificallywith the TRs could provide important clues to these processes and otherpotential functions of the receptors, such as regulation of cellproliferation (Halperin et al., Endocrinology 126:2321-2326, 1990). Inaddition, interacting proteins provide a means of controlling andmodulating thyroid hormone receptor function.

SUMMARY OF THE INVENTION

In a first aspect, the invention generally features a method fordetermining whether a test protein is capable of interacting with anuclear hormone receptor protein. The method involves: (a) providing ahost cell which contains (i) a reporter gene operably linked to aprotein binding site; (ii) a first fusion gene which expresses a firstfusion protein, the first fusion protein including a nuclear hormonereceptor protein covalently bonded to a binding moiety which is capableof specifically binding to the protein binding site; and (iii) a secondfusion gene which expresses a second fusion protein, the second fusionprotein including the test protein covalently bonded to a weak geneactivating moiety; and (b) determining whether the test proteinincreases expression of the reporter gene as an indication of itsability to interact with the nuclear hormone receptor protein.

In a preferred embodiment, the method further involves treating the hostcell with a ligand which binds the nuclear hormone receptor andidentifying a hormone-dependent interacting protein by its ability toincrease expression of the reporter gene only upon treatment of the cellby the ligand. In another preferred embodiment, the method furtherinvolves treating the host cell with a ligand which binds the nuclearhormone receptor and identifying a hormone-independent interactingprotein by its ability to increase expression of the reporter gene bothin the presence and in the absence of ligand treatment. In yet anotherpreferred embodiment, the method further involves treating the host cellwith a ligand which binds the nuclear hormone receptor and identifying aligand-sensitive interacting protein by its ability to increaseexpression of the reporter gene in the absence but not in the presenceof the ligand treatment. Preferably, the ligand is a thyroid hormone.

In other preferred embodiments, the weak gene activating moiety is thegene activating moiety of B42 or a gene activating moiety of lesseractivation potential; and the nuclear hormone receptor is a thyroidhormone receptor.

In a second aspect, the invention features a substantially purepreparation of a thyroid hormone receptor (TR)-interacting protein.Preferably, the TR-interacting protein is JL1 or JL2; includes an aminoacid sequence substantially identical to an amino acid sequence shown inany of FIGS. 2-28 (SEQ ID NOS: 1, 3, 6-30); and is derived from amammal, for example, a human.

In a related aspect, the invention features purified DNA (for example,cDNA) which includes a sequence encoding a TR-interacting protein,preferably encoding a human TR-interacting protein, for example, theTR-interacting proteins JL1 or JL2.

In other related aspects, the invention features a vector and a cellwhich includes a purified DNA of the invention; a purified antibodywhich specifically binds a TR-interacting protein of the invention; anda method of producing a recombinant TR-interacting protein involvingproviding a cell transformed with DNA encoding a TR-interacting proteinpositioned for expression in the cell; culturing the transformed cellunder conditions for expressing the DNA; and isolating the recombinantTR-interacting protein. The invention further features recombinantTR-interacting protein produced by such expression of a purified DNA ofthe invention.

In yet another aspect, the invention features a therapeutic compositionwhich includes as an active ingredient a TR-interacting protein of theinvention, the active ingredient being formulated in a physiologically-acceptable carrier. Such therapeutic compositions are useful in a methodof treating thyroid disorders in a mammal, involving administering thetherapeutic composition to the mammal in a dosage effective to increasethyroid function (in the case of hypothyroidism) or decrease thyroidfunction (in the case of hyperthyroidism).

As used herein, "reporter gene" is meant a gene whose expression may beassayed; such genes include, without limitation, lacZ, amino acidbiosynthetic genes, e.g. the yeast LEU2 gene, or the mammalianchloramphenicol transacetylase (CAT) gene. Reporter genes may beintegrated into the chromosome or may be carried on autonomouslyreplicating plasmids (e.g., yeast 2μ plasmids).

By "operably linked" is meant that a gene and a regulatory sequence(s)are connected in such a way as to permit gene expression when theappropriate molecules (e.g., transcriptional activator proteins orproteins which include transcriptional activation domains) are bound tothe regulatory sequence(s).

By a "binding moiety" is meant a stretch of amino acids which is capableof directing specific polypeptide binding to a particular DNA sequence(i.e., a "protein binding site"). LexA represents a preferred DNAbinding moiety in the invention. However, any othertranscriptionally-inert or essentially transcriptionally-inert DNAbinding domain may be substituted. The GAL4 DNA binding domainrepresents a somewhat less preferred DNA binding moiety for the systemdescribed herein.

By "weak gene activating moiety" is meant a stretch of amino acids whichis capable of weakly inducing the expression of a gene to whose controlregion it is bound. As used herein, "weakly" is meant below the level ofactivation effected by GAL4 activation region II (Ma and Ptashne, Cell48:847, 1987) and is preferably at or below the level of activationeffected by the B42 activation domain of Ma and Ptashne (Cell 51:113,1987). Levels of activation may be measured using any downstreamreporter gene system and comparing, in parallel assays, the level ofexpression stimulated by the GAL4- or B42-polypeptide with the level ofexpression stimulated by the polypeptide to be tested.

By "TR-interacting protein" is meant a polypeptide which directly orindirectly physically interacts with a thyroid hormone receptor in thein vivo protein interaction assay described herein. Such an interactionmay be thyroid hormone dependent or independent or may be thyroidhormone sensitive; it may also be transient in nature. Preferably, sucha polypeptide has an amino acid sequence which is at least 80%,preferably 90%, and most preferably 95% or even 99% homologous to theamino acid sequence of an interacting protein described herein (e.g.,JL1 or JL2) at the point of interaction with the thyroid hormonereceptor, or at least 80% and preferably 90% homologous overall. A"TR-interacting protein", as used herein, does not include any of theRXR proteins or Pit-1.

By "thyroid hormone" is meant T3, triac, or T4, and less preferablyreverse T3.

By "substantially pure" is meant a preparation which is at least 60% byweight (dry weight) the compound of interest, i.e., a TR-interactingprotein. Preferably the preparation is at least 75%, more preferably atleast 90%, and most preferably at least 99%, by weight the compound ofinterest. Purity can be measured by any appropriate method, e.g., columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

By "purified DNA" is meant DNA that is not immediately contiguous withboth of the coding sequences with which it is immediately contiguous(one on the 5' end and one on the 3' end) in the naturally occurringgenome of the organism from which it is derived. The term thereforeincludes, for example, a recombinant DNA which is incorporated into avector; into an autonomously replicating plasmid or virus; or into thegenomic DNA of a prokaryote or eukaryote, or which exists as a separatemolecule (e.g., a cDNA or a genomic DNA fragment produced by PCR orrestriction endonuclease treatment) independent of other sequences. Italso includes a recombinant DNA which is part of a hybrid gene encodingadditional polypeptide sequence.

By "substantially identical" is meant an amino acid sequence whichdiffers only by conservative amino acid substitutions, for example,substitution of one amino acid for another of the same class (e.g.,valine for glycine, arginine for lysine, etc.) or by one or morenon-conservative substitutions, deletions, or insertions located atpositions of the amino acid sequence which do not destroy the functionof the protein (assayed, e.g., as described herein). Preferably, such asequence is at least 80%, more preferably 90%, and most preferably 95%homologous to one of the sequences of FIGS. 2-28 (SEQ ID NOS: 1, 3,6-30). A "substantially identical" nucleic acid sequence codes for asubstantially identical amino acid sequence as defined above.

By "transformed cell" is meant a cell into which (or into an ancestor ofwhich) has been introduced, by means of recombinant DNA techniques, aDNA molecule encoding (as used herein) a TR-interacting protein.

By "positioned for expression" is meant that the DNA molecule ispositioned adjacent to a DNA sequence which directs transcription andtranslation of the sequence (i.e., facilitates the production of, e.g.,a TR-interacting protein).

By "purified antibody" is meant antibody which is at least 60%, byweight, free from the proteins and naturally-occurring organic moleculeswith which it is naturally associated. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight, antibody, e.g., TR-interacting protein-specificantibody. A purified TR-interacting protein antibody may be obtained,for example, by affinity chromatography using recombinantly-producedTR-interacting protein and standard techniques.

By "specifically binds" is meant an antibody which recognizes and bindsTR-interacting protein but which does not substantially recognize andbind other molecules in a sample, e.g., a biological sample, whichnaturally includes TR-interacting protein.

Other features and advantages of the invention will be apparent from thefollowing detailed description thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are first briefly described.

FIGS. 1A and 1B show a genetic selection in yeast for the isolation ofTR-interacting protein-encoding cDNAs. The LexA/TRβ chimeras bind to thelexA binding site (lexA op) upstream of the LEU2 gene. FIG. 1A showsthat, in cells expressing a fusion protein consisting of the B42transactivation (TA) domain fused to a protein that does not interactspecifically with the lexA/TR chimera, the LEU2 gene is not expressed,and the cells require supplemental leucine for growth. FIG. 1B showsthat, in cells expressing a TA fusion to a protein capable of bindingthe lexA/TR chimera, the TA domain is brought specifically to thepromoter LEU2 expression is increased, and the cells do not requiresupplemental leucine.

FIG. 2 shows the complete amino acid sequence of JL1 (SEQ ID NO:1),aligned with the recently identified S. cerevisiae transcriptionalcoactivator SUG1 (Swaffield et al., Nature 357:698-700, 1992) (SEQ IDNO:2). Identities and conservative substitutions are indicated. Theoverall sequence identity is 73%. The boxed and bold residues from 190to 197 (JL1) represent a potential ATP binding site that is conserved inall members of this family. The boxed residues from 45 to 66 (JL1) are aputative leucine zipper, extended by 1 heptad toward the N-terminus inthis full length sequence, which appears to be unique to JL1 and SUG1.The N-terminal portion of the JL1 sequence (1-49) was derived fromsubcloned PCR products corresponding to the 5' end of the JL1 mRNA.Independent clones with identical sequence were isolated using internalJL1 and vector primers with a HeLa cell cDNA library as template. Themethionine residue assigned as the start codon is preceded by a stopcodon only 9 nucleotides upstream.

FIG. 3A shows the amino acid sequence of JL2 (SEQ ID NO:3); the two LIMdomains are underlined and the consensus C/D and H residues are bold.This sequence represents the human portion of the fusion proteinisolated as an activator of the lexA/TRβ chimera.

FIG. 3B shows the alignment of the LIM domains of JL2 (SEQ ID NO:4) withthose of Lin11 (SEQ ID NO:5). These domains in both proteins includematches to all consensus positions; the overall sequence identity is35%.

FIG. 4 (SEQ ID NO:6) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S112a-.

FIG. 5 (SEQ ID NO:7) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S103a.

FIG. 6 (SEQ ID NO:8) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S203a.

FIG. 7 (SEQ ID NO:9) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S204b.

FIG. 8 (SEQ ID NO:10) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S205a.

FIG. 9 (SEQ ID NO:11) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S249a.

FIG. 10 (SEQ ID NO:12) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S351a.

FIG. 11 (SEQ ID NO:13) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S101a.

FIG. 12 (SEQ ID NO:14) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S223a.

FIG. 13 (SEQ ID NO:15) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S239a.

FIG. 14 (SEQ ID NO:16) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S410a.

FIG. 15 (SEQ ID NO:17) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S418a.

FIG. 16 (SEQ ID NO:18) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S419a.

FIG. 17 (SEQ ID NO:19) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S107a-.

FIG. 18 (SEQ ID NO:20) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S213a-.

FIG. 19 (SEQ ID NO:21) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S113a-.

FIG. 20 (SEQ ID NO:22) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S116a-.

FIG. 21 (SEQ ID NO:23) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S309a-.

FIG. 22 (SEQ ID NO:24) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S227b-.

FIG. 23 (SEQ ID NO:25) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S215a-.

FIG. 24 (SEQ ID NO:26) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S223a-.

FIG. 25 (SEQ ID NO:27) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S240a-.

FIG. 26 (SEQ ID NO:28) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S139a.

FIG. 27 (SEQ ID NO:29) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S110a-.

FIG. 28 (SEQ ID NO:30) shows a partial nucleic acid sequence and deducedamino acid sequence of the TR-interacting protein S243b.

FIG. 29 shows a Northern analysis of JL1 and JL2 expression in varioushuman tissues. Specifically, 2 μg of poly A⁺ mRNA from the indicatedtissues (H, heart; B, brain; Pl, placenta; Lu, lung; Li, liver; SM,skeletal muscle; K, kidney; Pa, pancreas; all obtained from Clontech,Palo Alto, Calif.) was hybridized to JL1 and JL2 probes by standardtechniques and washed at high stringency (see Ausubel et al., infra).Equivalent loading of RNA was verified by hybridization with a humanactin cDNA probe.

FIG. 30 shows the amino acid comparison of polypeptide 351a SEQ ID NO:12and a portion of BCL3 SEQ ID NO:31. Identical amino acids are indicated,and approximate positions of ankyrin repeats are underlined in BCL3. Thefirst ankyrin repeat of BCL3 in the comparison corresponds to the 4th of7 total in the full-length sequence.

There now follows a description of the use of an in vivo interactiontrap system for the isolation of proteins which physically associatewith thyroid hormone receptor and a description of exemplary interactingproteins (termed, TR-interacting proteins). This system may be usedgenerally to isolate proteins which interact with any nuclear hormonereceptor. Because the system has such general application for theisolation of nuclear hormone receptor-interacting proteins, this exampleis designed to illustrate, not limit, the invention.

DETAILED DESCRIPTION

Applicants have used an in vivo interaction trap system (developed inthe laboratory of Dr. Roger Brent) to identify and isolate proteins thatphysically interact with nuclear hormone receptors and, in particular,with the ligand binding domain of the rat receptor TRβ. This system,based on the modular nature of transcription factors, allows directgenetic selection for proteins capable of interacting with a desiredprotein.

In general, DNA encoding the desired protein is fused to DNA encodingthe C-terminus of a DNA binding domain, for example, the DNA bindingdomain ofthe bacterial repressor LexA protein, to generate a chimerictranscription factor, which can be tested for function in yeast. In theinstant case, a lexA/TR chimera consisting of intact lexA fused to thehinge, ligand binding, and C terminal (D, E and F) domains of TRβ wasfound to be completely unable to activate transcription in either thepresence or absence of T3 ligand. This lack of transcriptionalactivation by the lexA/TR chimera provided the basis for applicants'genetic selection. As shown in FIG. 1, a yeast strain in whichexpression of the LEU2 gene is dependent on binding of an activator toupstream lexA binding sites (i.e.,operators) is unable to grow in theabsence of added leucine when this chimera is expressed. However, ifsuch a strain expresses a second chimeric protein which includes arelatively weak transcriptional activation domain (e.g., the B42activation domain of Ma and Ptashne, Cell51:113, 1987) fused to aprotein capable of interacting specifically with lexA/TR, LEU2 geneexpression is activated, and leucine is not required for growth.

Using this system, a number of proteins which interact with thyroidhormonereceptor were isolated as follows. A plasmid cDNA library wasproduced by standard techniques from HeLa cell mRNA and hadapproximately 10⁶ original members. Each of these cDNA inserts was fusedto the B42 transcriptional activation domain (Ma and Ptashne, Cell51:113, 1987), andexpression of the fusion protein was placed under thecontrol of the inducible yeast GAL10 promoter. In addition to the B42activating domain, this expression construct also carried, amino tocarboxy terminal, an ATG for protein expression, an optional nuclearlocalization sequence, and an optional epitope tag for rapidimmunological detection of fusion protein synthesis. The plasmid alsoincluded replication origins for yeast and E. coli as well as selectablemarkers for both.

The fusion protein library was introduced into a yeast strain thatexpressed the lexA/TRβ chimera and also contained two reporter genes:alexAop/LEU2 selection construct and a lexAop/β-galactosidase indicatorconstruct. Approximately 10⁷ initial transformants were generated undernonselective conditions, representing a several fold redundancy relativeto the original number of clones in the library. Thesetransformants wererecovered and replated under selective (leu⁻) conditions in the presenceor absence of thyroid hormone; based on the results of a functionalanalysis of intact TRs in yeast (Privalsky et al.,Cell 63:1277-1286,1990), a high concentration of triac (10⁻⁵ M) was added directly to theplates. A number of leucine-independent colonies that containedcandidate TR-interacting cDNAs were obtained under both conditions.

The specificity of an interaction between TR and a candidateTR-interactingprotein can be checked in several ways. For example,clones which do not activate expression of the lexA/β-galactosidaseconstruct can be eliminated. These clones generally include yeastmutants that activate theLEU2 promoter or mammalian cDNAs that activateby some means other than through the lexA binding sites. Since theexpression of the cDNA library fusion protein is under the control of aninducible promoter, the dependence of reporter gene expression on thischimera can also be tested by this criterion.

cDNA library plasmids were recovered from those yeast strains whichpassed the above tests. Each was reintroduced into the original lexA/TRstrain, and their ability to specifically activate expression wasconfirmed. This step was included because yeast transformants frequentlycontain one or more plasmids, in addition to the one that allowssurvival under the selective conditions. To confirm their specificityfor TRβ interaction, the rescued plasmids were also introduced intostrains containing other lexA chimeras generated in Dr. Brent'slaboratory; these included lexA/myc and lexA/cdc2. All of the cloneswere found to be specific for TRβ by this criterion. cDNA clones thatpassed all of the above tests were concluded to encode proteins thatcould specifically interact with the lexA/TR chimera.

Based on restriction mapping, these clones were sorted into distinctclasses. Members of each class were sequenced across the fusion junctionwith the transcriptional activation domain. Sequences of many of theseproteins are shown in FIGS. 2-28 (SEQ ID NOS: 1, 3, 6-30).

Although some clones have shown no significant similarities in searchesof the sequence databases, most have shown some relationship to knownproteins. As described below, two classes showed strong matches overlimited domains to nuclear transcription factors. One clone unexpectedlyappeared to encode a fragment of the human clathrin heavy chain. Sincecell biology considerations argue that TRβ is quite unlikely to have abiologically relevant interaction with clathrin in mammalian cells, itcan be assumed that the sensitivity of the selection system allowsisolation of fragments of proteins that show some affinity for TR basedsolely on simple chemical interactions. Interacting proteins of thissort are useful for the production of peptides which interfere withthyroid hormone receptor function (see below).

Because RXRβ interacts with TR and is expressed in HeLa cells (Liedetal., Cell 68:377-395, 1992), RXR fusions would be expected to activatereporter gene expression and be isolated in this selection. To test thisprediction, a fragment encoding the RXRα hinge and ligand bindingdomains was inserted in frame into the transcriptional activation domainfusion vector used to generate the original cDNA library. As expected,this RXR fusion construct allowed the lexA/TR tester strain, but notstrains expressing other lexA chimeras, to survive in the absence ofleucine and also activated expression of the lexA/β-galactosidasereporter gene (see Table 2). RXR, however, was not identified in theoriginal screen. This is most likely explained by the fact that,although the original library was large, it was extensively amplified,which can decrease representation of rare cDNAs. Moreover, the fusion tothe transcriptional activation domain must be in the correct frame andmay be functional if the fusion occurs in only a relatively limitednumber of positions. Since members of the nuclear hormone receptorsuperfamily are generally expressed at extremely low levels, it is mostlikely that appropriate RXR clones were simply not present in theamplified library originally screened.

Unexpectedly, nearly all of the lexA/TR interacting cDNAs showed verystrong dependence on hormone for activation. Two proteins, JL1 and JL2,which were isolated in the initial selection in the presence of triac,both interacted with the lexA/TR chimera much more strongly when triacwaspresent, as judged by level of expression of β-galactosidase. Thishormone⁺ group constituted the majority of isolated clones (>10different classes), although there were a smaller number in a hormone⁻group that interacted only when triac was absent. These classes areshown in Table 1.

                  TABLE 1    ______________________________________    Class 1    JL1;       homologous to HIV/TAT interacting               proteins MSS1 (Nature 357: 700-702,               1992), and to yeast SUG1 (Nature               357: 698-700, 1992)    JL2;       contains LIM domain (Nature 344: 876-879,               1992)    112a-;     no significant homology to any known               gene in current databank    103a;      homology to homeobox protein CUT (Nature               333: 629-635, 1988)    203a;      homologous to bovine               phosphatidylethanolamine-binding protein               (EUR. J. BIOCHEM. 166, 333-338, 1987)    204b;      homologous to kinesin-related protein               (Mol. Cell. Biol. 11: 3395-3398, 1991)    205a;      no significant homology to any known               gene in current databank    249a;      no significant homology to any known               gene in current databank    351a;      homology to BCL3 (Cell 60: 991-997, 1990)    101a;      homology to GRP94 (J. Biol. Chem. 262:               8875-8883, 1987)    223a;      no significant homology to any known               gene in current databank    239a;      contains HMG box (Nature 357: 282-283,               1992)    410a;      contains SH3 domain (Science               252: 668-674, 1991)    417a;      identical to human dUTP pyrophosphatase               (Proc. Natl. Acad. Sci. U.S.A.               89: 8020-8024, 1992)    418a;      no significant homology to any known               gene in current databank    419a;      homology to yeast N-myristoyltransferase               (Science 243: 796-800, 1989)    Class 2    107a-;     homologous to rat clathrin heavy chain               (Proc. Natl. Acad. Sci. U.S.A.               84: 8805-8809, 1987)    213a-;     no significant homology to any known               gene in current databank    113a-;     no significant homology to any known               gene in current databank    116a-;     no significant homology to any known               gene in current databank    309a-;     homologous to mouse perform (Proc.               Natl. Acad. Sci. U.S.A. 86: 247-251,               1989)    227b-;     homologous to mitochondrial hsp70 (DNA               8: 233-243, 1989)    224a-;     identical to human ferritin heavy chain               (EMBO J. 3: 23-27, 1984)    312b-;     identical to human hnRNP C1/2 (Proc.               Natl. Acad. Sci. U.S.A. 86: 9788-9792,               1989)    215a-;     no significant homology to any known               gene in current databank    223a-;     homology to (2'-5') oligoadenylate               synthetase (EMBO J. 4: 2249-2256, 1985)    240a-;     no significant homology to any known               gene in current databank    Class 3    139a;      homology to possible transcription               factor VAC1 (J. Biol. Chein. 267: 618-623               1992)    110a-;     no significant homology to any known               gene in current databank    ______________________________________

The fact that virtually all of the isolated clones were specific for onehormone state or the other was surprising.

The genetic properties of sample TR-interacting proteins and RXR fusionproteins are summarized in Table 2.

                  TABLE 2    ______________________________________                  Lex A fusion                                        lexA/    TA fusion       lexA      lexA/TR   c-myc    ______________________________________    B42 (vector)                -T3     leu.sup.-, W                                  leu.sup.-, W                                          leu.sup.-, W                +T3     leu.sup.-, W                                  leu.sup.-, W                                          leu.sup.-, W    JL1/JL2     -T3     leu.sup.-, W                                  leu.sup.-, W                                          leu.sup.-, W                +T3     leu.sup.-, W                                  leu.sup.+, B                                          leu.sup.-, W    RXR         -T3     leu.sup.-, W                                  leu.sup.+, B                                          leu.sup.-, W                +T3     leu.sup.-, W                                  leu.sup.+, B                                          leu.sup.-, W    ______________________________________

Each of the strains shown in Table 2 contained both the lexAop/LEU2 andthelexAop/β-galactosidase reporter constructs, along with the indicatedtranscriptional activation (TA) domain fusion proteins; the cDNA cloningvector expressed the B42 transcriptional activation domain alone (Ma andPtashne, Cell 51:113-119, 1987). Cells containing the indicated TAfusion proteins were transformed with each of the indicated lexA fusionvectors, and phenotypes were tested under various conditions. ±T3indicates the presence or absence of 10⁻⁵ M triac in the plates(Privalsky et al., Cell 63:1277-1286, 1990); leu.sup.∓ denotes theability of the transformed cells to grow on plates lacking leucine; W/Bindicates formation of white or blue colonies on indicator platescontaining the indicator X-gal. As expected, the activation conferred bythe JL1, JL2, and RXR fusion proteins was dependent on the specificinduction of the GAL10 promoter that controls their expression.

JL1 and JL2

The largest class of lexA/TR interacting cDNAs (17 individual isolates)encoded JL1 (also called thyroid hormone receptor-interacting protein 1,or TRIP1). All of the members of the class exhibited the propertiessummarized above, although some variations in the levels of expressionof β-galactosidase in the presence or absence of hormone was observedfor clones that varied in position of the junction to the B42transactivation domain. JL1 is quite similar to several previouslyidentified proteins, particularly TBP1, as indicated in FIG. 2. Thefunctions of this family of proteins are diverse: TBP1 is a nuclearprotein that has a poorly understood but apparently important role intranscriptional regulation of HIV (Nelbock et al., Science248:1650-1653, 1990), while the mammalian protein VCP (Koller andBrownstein, Nature 325:542-545, 1987) and its apparent yeast homologCDC48 (Frohlich et al., J. Cell. Biol. 114:443-453, 1991) arecytoplasmic proteins of unknown function. TBP1 was isolated by usinglabeled HIV TAT protein to screen a lambda gt11 expression library andhas been found to interact directly with that important viral regulatorbut not with DNA. Although initially described as an inhibitor of TATfunction in cotransfections, a more recent report indicates that TBP1may act to stimulate TAT activity and may have a direct transcriptionalactivation function in its own right (Rosen, Abstract. Cold SpringHarbor Symp. Quant. Biol. 57:267, 1992). On these grounds, TBP could beconsidered a candidate transcriptional coactivator. JL1 is even morehomologous to SUG1 (74%, see FIG. 2), a yeast gene recently isolated asa suppressor of a defective version of theGAL4 activator (Swaffield etal., Nature 357:698, 1992). By genetic analysis, SUG1 appears to be acoactivator capable of specifically interacting with GAL4, and JL1similarly encodes a thyroid hormone-dependent coactivator protein.Functionally, they are at least partially homologous, with expression ofJL1 able to rescue a SUG1 temperature-sensitive lethal mutant in a yeastsystem in a similar manner to wild-type SUG1 (Swaffield et al.,manuscript submitted). This interchangeability indicates that the SUG1and JL1 transcriptional function has been highly conserved, most likelywithin the conserved ATPase-containing domain common to the superfamily.Thus, they are likely to bind the same activation domains and exerttranscriptional control in asimilar manner.

JL2, encoded by a single recovered cDNA, includes two copies of the LIMdomain originally identified as a conserved motif in three putativetranscription factors: Lin-11 (Freyd et al., Nature 344:876-879, 1990),Isl-1 (Karlsson et al., Nature 344:879-882, 1990) and Mec-3 (Way, andChalfie, Cell 54:5-16, 1988). In the context of endocrine control ofgene expression, isl-1 is particularly interesting since it is anactivator of the insulin enhancer. It is expressed in both developingand mature islet cells and is thought to be involved in the initialdifferentiation of the islet cells, in addition to its presumed role inregulating insulin expression. Isl-1 is also expressed in a subset ofneurons in the adult and, recently has been shown to be expressed atvery early stages of embryonic motor neuron differentiation. The patternof this early expression suggests that isl-1 may play a primary role inthe initial determination of motor neuron cell fate in response toinductive signals from the notochord and floor plate (Ericson et al.,Science 256:155-1560, 1992). Consistent with this possibility, lin-11and mec-3 are both C. elegans developmental regulators, associated withcell lineage determination in mechanosensory neurons and a vulvalprecursor cell, respectively.

Lin-11, isl-1, and mec-3 contain a homeobox-type DNA binding domain inaddition to two copies of the LIM domain, as do other recentlyidentified members of this family (see, e.g., Cohen et al., Genes Dev.6:715-729, 1992; Taira et al., Genes Dev. 6:356-366, 1992). However, ahomeodomain isabsent in a three related LIM domain-containing proteinscalled rhombotins 1-3, at least two of which are the products ofputative oncogenes (Rosen, Abstract, Cold spring Harbor Symp. Quant.Biol. 57:267, 1992). The LIM domain consensus sequence containsconserved cysteine and histidine residues, and it has recently beendemonstrated that at least the lin-11 version binds metal ions (2 atomsof Zn and 4 of Fe; Li et al., Proc. Natl. Acad. Sci. U.S.A. 88:9210,1991). As indicated in FIG. 3, JL2 has a good match with the LIMconsensus in lin-11; it does not, however, includea homeobox. In thisregard, JL2 appears to be more like the rhombotins thanit is like thetranscription factors lin-11, isl-1, and mec-3.

An initial determination of the pattern of expression of JL1 and JL2 hasbegun. As indicated in FIG. 29, the approximately 2.1 kb JL1 mRNA isexpressed at various levels in all the human tissues examined. Theslightly smaller 1.8 kb JL2 mRNA is expressed in a somewhat narrowerrangeof tissues. Based on the amount of time required to visualize thebands, both mRNAs are present at very low levels, consistent with aregulatory role. As judged by exposure time, the JL1 mRNA appears to beexpressed at an approximately 6 fold higher level than that of JL2, aswould be expected from the higher number of JL1 clones isolated.

S351a

The isolated polypeptide 351a was found to have some homology (about 40%identity at the amino acid level) to BCL3. The BCL3 gene product ischaracterized by seven 30 amino acid ankyrin repeats (Ohno et al., Cell60:991-997, 1990), so named because of their initial identification intheerythrocyte membrane protein ankyrin. There are now many examples ofrelated proteins, which share the repeated structure consisting of aloosely conserved, approximately 30 amino acid motif (the ankyrinrepeat).These related proteins have diverse functions, but one subgroup,including BCL3, IκB, and others have specific functions in theregulation of transcription. These proteins bind specifically to thefamily of related proteins that form a dimeric transcription factorgenerically known as NFκB. The interaction of IκB with NFκB inhibits itsability to activate transcription of target genes because the complex isretained in the cytoplasm. This retention is possibly due to IκB bindingto and masking NFκB's nuclear localization signals. The interaction withBCL3, in contrast, apparently occurs in the nucleus and leads to astimulation of transcriptional activity by unknown mechanisms. Thisfamily of proteins thus appears to have a modulatory effect ontranscription, which allows regulation of a variety of gene productsinvolved in numerous cellular responses. Hence, members of this subgroupcan have either inhibitory or stimulatory effects on transcription, anditis unclear what function in this regard the 351a peptide has in itsinteractions with TR. FIG. 30 shows the similarity between 351a and therelevant portion of BCL3. The ankyrin repeats of BCL3 are underlined.The similarity is clearly greatest over the N-terminal portion of thesequenceshown, in the stretch which corresponds to the 4th of the 7repeats in BCL3. Overall, the relationship is not particularly strong,and may account for the observation the BCL3 does not interact witheither TR or RXR in the interaction trap of this invention, while 351adoes. 351a clearly has a ligand dependent TR and RXR interactionfunction not shared by its closest relative within the family ofproteins containing ankyrin repeats.

Experiments in yeast have shown that although both the lexA-TR chimerausedin the interaction trap and TR alone activate transcription in yeastvery poorly in the presence or absence of thyroid hormone, coexpressionof the TR heterodimer partner RXR restores hormone dependenttranscriptional activation of both (on lexA operators and TR bindingsites, respectively).Adding the lexA-351a construct to the intact TR+RXRstrongly inhibits this latter activation. This inhibition could be aconsequence of a direct inhibition associated with 351a binding, orcould be an indirect effect associated with the fact that the lexA-351achimera is missing essential sequences necessary for a co-stimulatoryfunction analogous to that of BCL3. In an additional series ofexperiments, lexA-351a alone was also found to be transcriptionallyinactive in yeast. However, coexpression of intact TR causes lexA-351ato become a T3-dependent transcriptional activator. This indicates thatthe interaction of 351a with TR does not result in a complex that isinherently inactive, resolution of the larger question of whether thenative 351-TR interaction is stimulatory or inhibitory will requireanalysis of the function of the full length 351a protein.

From a practical point of view, the inhibitory effect of the lexA-351achimera provides a direct demonstration of a potentially useful functionof the truncated protein, namely inhibition of TR function. Liposomes orother delivery systems known in the art could be employed to deliverthis truncated protein or the minimum biologically active fragmentthereof for therapeutic uses. The direct demonstration of the negativeeffect providesa clear demonstration of the general concept of using351a to block TR action. 351a could provide a therapeutically usefulantagonist of TR function analogous to, for instance, the anti-steroideffects of RU486, a drug that inhibits transcription in members of thenuclear hormone receptor family. The mechanism of action of RU486 isdistinct from 351a, and does not exert effects on thyroid hormonereceptors, and thus the inhibitory effect of 351a on transcriptionalregulation in the thyroid hormone receptor system is of potentiallymajor significance.

To determine whether a TR-interacting protein has a positive or anegative effect on TR function, cotransfections of the TR-interactingprotein expression vector and a TRβ or TRα expression vector are carriedout by standard techniques, preferably, in a host cell line that doesnot express significant levels of the TR-interacting protein (see, e.g.,Ausubel et al., Current Protocols in Molecular Biology, John Wiley Sons,1989). A TR-interacting protein which acts as a positive regulator(e.g., a coactivator), is indicated by increased TR activity in such anassay. Conversely, a TR-interacting protein which acts as a negativeregulator is indicated by reduced TR activity. Cotransfection assays ofthis sort are generally described in Ausubel et al. (supra).

In one particular example, the TR-interacting protein-encoding cDNA isinserted into the CDM8 vector (Seed, B., Nature 329:840, 1987), andincreasing doses of this plasmid are cotransfected with a TRβ expressionvector (Brent et al., J. Biol. Chem. 264:178, 1989) plus one ofseveraldifferent reporter genes containing various T3REs linked, e.g., to theherpes virus thymidine kinase (TK) gene (Brent supra). In thesetransfections, the level of total expression vector is maintained at aconstant level by addition of CDM8, as necessary. To control forvariations in transfection efficiency and for effects of theTR-interacting protein on the TK promoter, transfections also includepTKGH (Selden et al., Moll. Cell. Biol. 6:3173, 1986), a plasmid whichdirects expression of human growth hormone under the control of the sameTK promoter. As controls for regulatory effects, Pit-1, c-fos and c-junmay also be cotransfected with TRβ and the T3RE reporters.

Since the relative and absolute levels of expression of TRβ and itspotential partners may be crucial for observation of any effect, anegative result is first confirmed at a variety of doses of each vector.Several cell lines are also examined. If however, no evidence for aspecific effect of a TR-interacting protein on TR function is observedafter these steps, it will be concluded that the interaction with TRβislikely to be an artifact of the sensitivity of the genetic selectionoriginally used to isolate them.

If, on the other hand, the TR-interacting protein alters TR function,the specificity of the effect is examined. Simple cotransfections of theTR-interacting protein expression vector with RSVCAT or TKCAT vectors isused to confirm that any negative effect is not a consequence ofsquelching (Ptashne and Gann, Nature 346:329-331, 1990). Cotransfectionsof appropriate reporters with the TR-interacting protein expressionvectors plus vectors expressing TRα, the RARs, VDR, GR, ER or othersmayalso be carried out.

The portions of any particular TR-interacting protein required forfunctional interaction may be determined initially by standard deletionanalysis, with mutant proteins tested by the above cotransfection assay.The results of such mapping may be confirmed and extended by testing theeffect of the same mutations on the lexA/TR dependent activation ofexpression in yeast, and by the following biochemical interactionassays.

To determine directly whether a TR-interacting protein can interact withthyroid hormone receptor, antiserum directed against one of thepotential partners is tested for its ability to coimmunoprecipitate theother. This may be assayed directly using bacterially-produced TRproteins and antiserum or monoclonal antibodies that recognize someregion of the TRβ protein. In one particular example of such an assay,in vitro translated, ³⁵ S labeled TR-interacting protein is mixed withTRβ protein in the presence or absence of T3, and the mixture isimmunoprecipitated with an antiserum that recognizes the N-terminus oftheTR. Similarly labeled RXRβ protein, which is known to interactstrongly with TRβ in such procedures, is used as a positive control. Theimmunoprecipitated material is resolved by SDS PAGE, and the presence ofthe TR-interacting protein or RXR in such immunoprecipitates is assessedby autoradiography. The observation of T3- dependentcoimmunoprecipitation of the potential TR binding proteins with the TRprovides strong evidence for a direct interaction with the receptor. Ageneral description of in vitro translation of proteins is described inHope and Struhl, Cell 43:177-188, 1985. Labelling proteins with ³⁵S,production of antibodies (including monoclonal antibodies), andimmunoprecipitation procedures are described in Ausubel (infra).

Lack of such a coimmunoprecipitation may suggest that the interaction ofa particular protein with TR is too transient to be detected by thisapproach. This can be tested by addition of various crosslinkingreagents to the binding reactions, as described in the analysis of theinteractionsof GR with AP-1, for example (Yang-Yen et al., Cell62:1205-1215, 1990). Itis important to control for the variety ofartifactual associations that may complicate interpretation of suchstudies. If crosslinking does not reveal an interaction between aTR-interacting protein and TRβ, even in the presence of extracts thatmight supply additional cofactors required, it may be that theirinteraction in yeast is artifactual.

Truncated versions of TR-interacting proteins can also be tested usingthismethod to identify specific portions of each protein required for TRinteraction. This is of particular importance from the point of view ofpotential pharmacologic intervention with the interaction, since suchfragments may facilitate the production of specific inhibitors of TRfunction.

TR-Interacting Proteins and Antibodies

Polypetide Expression

In general, polypeptides according to the invention may be produced bytransformation of a suitable host cell with all or part of aTR-interacting protein-encoding cDNA fragment (e.g., the cDNA describedabove) in a suitable expression vehicle.

Those skilled in the field of molecular biology will understand that anyofa wide variety of expression systems may be used to provide therecombinantprotein. The precise host cell used is not critical to theinvention. The TR-interacting protein may be produced in a prokaryotichost (e.g., E. coli) or in a eukaryotic host (e.g., Saccharomycescerevisiae or mammaliancells, e.g., COS 1, NIH 3T3, or HeLa cells). Suchcells are available from a wide range of sources (e.g., the AmericanType Culture Collection, Rockland, Md.; also, see, e.g., Ausubel et al.,Current Protocols in Molecular Biology, John Wiley Sons, New York,1989). The method of transformation or transfection and the choice ofexpression vehicle will depend on the host system selected.Transformation and transfection methods are described, e.g., in Ausubelet al. (Current Protocols in Molecular Biology, John Wiley Sons, NewYork, 1989); expression vehicles may be chosen from those provided,e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et al.,1985, Supp. 1987).

One preferred expression system is the mouse 3T3 fibroblast host celltransfected with a pMAMneo expression vector (Clontech, Palo Alto,Calif.). pMAMneo provides: an RSV-LTR enhancer linked to adexamethasone-inducible MMTV-LTR promotor, an SV40 origin of replicationwhich allows replication in mammalian systems, a selectable neomycingene,and SV40 splicing and polyadenylation sites. DNA encoding aTR-interacting protein would be inserted into the pMAMneo vector in anorientation designed to allow expression. The recombinant TR-interactingprotein wouldbe isolated as described below. Other preferable host cellswhich may be used in conjunction with the pMAMneo expression vehicleinclude COS cells and CHO cells (ATCC Accession Nos. CRL 1650 and CCL61, respectively).

Alternatively, a TR-interacting protein is produced by astably-transfectedmammalian cell line. A number of vectors suitable forstable transfection of mammalian cells are available to the public,e.g., see Pouwels et al. (supra); methods for constructing such celllines are also publicly available, e.g., in Ausubel et al. (supra). Inone example, cDNA encoding the TR-interacting protein is cloned into anexpression vector which includes the dihydrofolate reductase (DHFR)gene. Integration of the plasmid and, therefore, the TR-interactingprotein-encoding gene into the host cell chromosome is selected for byinclusion of 0.01-300 μM methotrexate in the cell culture medium (asdescribed in Ausubel et al., supra). This dominant selection can beaccomplished in most cell types. Recombinant protein expression can beincreased by DHFR-mediated amplification of the transfected gene.Methods for selecting cell lines bearing gene amplifications aredescribed in Ausubel et al. (supra); such methods generally involveextended culture in medium containing gradually increasing levels ofmethotrexate. DHFR-containing expression vectors commonly used for thispurpose include pCVSEII-DHRF and pAdD26SV(A) (described in Ausubel etal., supra). Any of the host cells described above or, preferably, aDHFR-deficient CHO cell line (e.g., CHO DHFR⁻cells, ATCC Accession No.CRL 9096) are among the host cells preferred for DHFR selection of astably-transfected cell line or DHFR-mediated gene amplification.

Once the recombinant TR-interacting protein is expressed, it isisolated, e.g., using affinity chromatography. In one example, ananti-TR-interacting protein antibody (e.g., produced as describedherein) may be attached to a column and used to isolate theTR-interacting protein. Lysis and fractionation of TR-interactingprotein-harboring cellsprior to affinity chromatography may be performedby standard methods (see,e.g., Ausubel et al., supra). Alternatively, aTR-interacting protein fusion protein, for example, a TR-interactingprotein-maltose binding protein, a TR-interactingprotein-β-galactosidase, or a TR-interacting protein-trpE fusionprotein, may be constructed and used for TR-interacting proteinisolation (see, e.g., Ausubel et al., supra; New England Biolabs,Beverly, Md.).

Once isolated, the recombinant protein can, if desired, be furtherpurified, e.g., by high performance liquid chromatography (see, e.g.,Fisher, Laboratory Techniques In Biochemistry And Molecular Biology,eds.,Work and Burdon, Elsevier, 1980).

Polypeptides of the invention, particularly short TR-interacting proteinfragments, can also be produced by chemical synthesis (e.g., by themethods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 ThePierce Chemical Co., Rockford, Ill.).

These general techniques of polypeptide expression and purification canalso be used to produce and isolate useful TR-interacting proteinfragments or analogs (described herein).

Anti-TR-Interacting Protein Antibodies

Human TR-interacting protein (or immunogenic fragments or analogues) maybeused to raise antibodies useful in the invention; such polypeptidesmay be produced by recombinant or peptide synthetic techniques (see,e.g., Solid Phase Peptide Synthesis, supra; Ausubel et al., supra). Thepeptides may be coupled to a carrier protein, such as KLH as describedin Ausubel et al, supra. The KLH-peptide is mixed with Freund's adjuvantand injected into guinea pigs, rats, or preferably rabbits. Antibodiesmay be purified by peptide antigen affinity chromatography.

Monoclonal antibodies may also be prepared using the TR-interactingproteins described above and standard hybridoma technology (see, e.g.,Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol.6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling etal., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y.,1981;Ausubel et al., supra).

Once produced, polyclonal or monoclonal antibodies are tested forspecific TR-interacting protein recognition by Western blot orimmunoprecipitation analysis (by the methods described in Ausubel etal., supra). Antibodies which specifically recognize a TR-interactingprotein are considered to beuseful in the invention; such antibodies maybe used, e.g., in an immunoassay to monitor the level of TR-interactingprotein produced by a mammal or to determine the subcellular location ofany of these thyroid hormone receptor modulatory proteins.

Preferably, antibodies of the invention are produced using fragments oftheTR-interacting protein which lie outside highly conserved regions andappear likely to be antigenic, by criteria such as high frequency ofcharged residues. In one specific example, such fragments are generatedbystandard techniques of PCR and cloned into the PGEX expression vector(Ausubel, F. M. et al., Current Protocols in Molecular Biology (GreenePub. Assoc., New York, 1992). Fusion proteins are expressed in E. coliandpurified using a glutathione agarose affinity matrix as described in(Ausubel, F. M. et al., Current Protocols in Molecular Biology (GreenePub. Assoc., New York, 1992)). To attempt to minimize the potentialproblems of low affinity or specificity of antisera, two or three suchfusions are generated for each protein, and each fusion is injected intotwo rabbits. Antisera are raised by injections in a series including atleast three booster injections. This approach has been successfully usedby applicants to generate antibodies capable of discriminating betweenthedifferent TR isoforms.

Antisera is cleared of anti-GST antibodies using GST immobilized on aglutathione column, and the antisera are checked by ELISA for titer andspecificity, using GST fusion proteins as controls. Antisera is alsochecked for its ability to immunoprecipitate in vitro translatedTR-interacting proteins or control proteins, such as Pit-1 or RARα.Western blots of total or nuclear vs. cytoplasmic fractionated HeLa cellproteins are also probed with the antisera to assess specificity and tocharacterize subcellular compartmentalization. In these and otherimmunologic assays, specificity is confirmed by the specific competitionwith the GST fusion protein.

Once the specificity of an antiserum is confirmed, it may be used in anystandard indirect immunofluorescence procedure to determine thesubcellular distribution of the TR-interacting protein in a particularcell type. Based on their similarity to nuclear transcriptionalregulatorsand their interaction with TRs, TR-interacting proteins arelikely to be nuclear localized.

Use

The proteins described herein interact with thyroid hormone receptor andare thus likely to mediate or modulate TR function. Because of theireffects on thyroid receptor activity, such proteins (or peptides derivedfrom these proteins, particularly, short peptides which are capable ofTR interaction), may facilitate the production of pharmacologicmodifiers of receptor function.

In particular, TR-interacting proteins of the invention which positivelyregulate TR function in vivo or in vitro (e.g., as assayed incotransfections as described above) may be used to produce therapeuticpeptides which include a TR interaction domain but which lack a TRactivity-enhancing domain, for example, a domain which interacts withthe transcriptional apparatus; the efficacy of such peptides may also,e.g., as assayed as described above. Such peptides would bind TR,interfering with receptor binding by the native TR-interacting protein,and thereby reducing TR activity. Peptides of this sort would be usefulin the treatment of hyperthyroidism.

Conversely, interacting peptides derived from TR-interacting proteinswhichnegatively regulate TR function, as assayed in vivo or in vitro(again, e.g., by the assays described above) may be used to producetherapeutic peptides which block the normal interaction between thereceptor and the negatively acting TR-interacting protein. Thesepeptides may similarly be administered to a mammal to treat thyroiddisorders.

Such therapeutic polypeptides of the invention may be administered byany appropriate route, e.g., intravenously, at a dosage which iseffective to increase or decrease thyroid function. Treatment may berepeated as necessary for alleviation of disease symptoms.

The polypeptides of the invention are also useful for identifying thosecompartments of mammalian cells which contain proteins important to thefunction of the thyroid hormone receptor. Antibodies specific for aparticular TR-interacting protein (or any nuclear hormonereceptor-interacting protein) may be produced as described above. Thenormal subcellular location of the protein is then determined either insitu or using fractionated cells by any standard immunological orimmunohistochemical procedure (see, e.g., Ausubel et al., supra;Bancroft and Stevens, Theory and Practice of Histological Techniques,Churchill Livingstone, 1982).

Antibodies specific for TR-interacting proteins also find diagnostic useinthe detection or monitoring of thyroid disorders. Levels of aTR-interacting protein in a sample may be assayed by any standardtechnique. For example, its expression may be monitored by standardNorthern blot analysis or may be aided by PCR (see, e.g., Ausubel etal., supra; PCR Technology: Principles and Applications for DNAAmplification, ed., H. A. Ehrlich, Stcokton Press, New York). Thesetechniques are enabled by the provision of the TR-interacting proteinsequences describedherein. Alternatively, standard immunological orimmunohistochemical procedures (e.g., those described above) may also beused with the antibodies described herein for TR-interacting proteindetection.

Other Embodiments

In other embodiments, the invention includes any protein which issubstantially homologous to a human TR-interacting protein (FIGS. 2-28,SEQ ID NOS: 1, 3, 6-30); such homologs include other substantially purenaturally occurring mammalian TR-interacting protein proteins as well asallelic variants; natural mutants; induced mutants; proteins encoded byDNA that hybridizes to the TR-interacting protein sequence of any ofFIGS.2-28 (SEQ ID NOS: 1, 3, 6-30) under high stringency conditions orlow stringency conditions (e.g., washing at 2× SSC at 40° C. witha probelength of at least 40 nucleotides); and polypeptides or proteinsspecifically bound by antisera directed to a TR-interacting protein,especially by antisera to the TR binding domain of the TR-interactingprotein. The term also includes chimeric polypeptides that include aTR-interacting protein fragment.

The invention further includes analogs of any naturally occurringTR-interacting protein. Analogs can differ from the naturally occurringTR-interacting protein by amino acid sequence differences, bypost-translational modifications, or by both. Analogs of the inventionwill generally exhibit at least 80%, more preferably 90%, and mostpreferably 95% or even 99%, homology with all or part of a naturallyoccurring TR-interacting protein sequence. The length of comparisonsequences will be at least 8 amino acid residues, preferably at least 24amino acid residues, and more preferably more than 35 amino acidresidues.Modifications include in vivo and in vitro chemicalderivatization of polypeptides, e.g., acetylation, carboxylation,phosphorylation, or glycosylation; such modifications may occur duringpolypeptide synthesis or processing or following treatment with isolatedmodifying enzymes. Analogs can also differ from the naturally occurringTR-interacting protein by alterations in primary sequence. These includegenetic variants, both natural and induced (for example, resulting fromrandom mutagenesis by irradiation or exposure to ethanemethylsulfate orby site-specific mutagenesis as described in Sambrook, Fritsch andManiatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press,1989, hereby incorporated by reference; or Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, 1989, herebyincorporated by reference). Also included are cyclized peptidesmolecules and analogs which contain residues other than L-amino acids,e.g., D-amino acids or non-naturally occurring or synthetic amino acids,e.g., β or γ amino acids.

In addition to full-length polypeptides, the invention also includesTR-interacting protein fragments. As used herein, the term "fragment",means at least 10 contiguous amino acids, preferably at least 30contiguous amino acids, more preferably at least 50 contiguous aminoacids, and most preferably at least 60 to 80 or more contiguous aminoacids. Fragments of TR-interacting proteins can be generated by methodsknown to those skilled in the art or may result from normal proteinprocessing (e.g., removal of amino acids from the nascent polypeptidethatare not required for biological activity or removal of amino acidsby alternative mRNA splicing or alternative protein processing events).

Preferable fragments or analogs according to the invention are thosewhich facilitate interaction of the peptide with a thyroid hormonereceptor.

Other embodiments are within the following claims.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 31    (2) INFORMATION FOR SEQ ID NO: 1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 406    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: Not Relevant    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:    MetAlaLeuAspGlyProGluGlnMetGluLeuGluGluGlyLysAla    151015    GlySerGlyLeuArgGlnTyrTyrLeuSerLysIleGluGluLeuGln    202530    LeuIleValAsnAspLysSerGlnAsnLeuArgArgLeuGlnAlaGln    354045    ArgAsnGluLeuAsnAlaLysValArgLeuLeuArgGluGluLeuGln    505560    LeuLeuGlnGluGlnGlySerTyrValGlyGluValValArgAlaMet    65707580    AspLysLysLysValLeuValLysValHisProGluGlyLysPheVal    859095    ValAspValAspLysAsnIleAspIleAsnAspValThrProAsnCys    100105110    ArgValAlaLeuArgAsnAspSerTyrThrLeuHisLysIleLeuPro    115120125    AsnLysValAspProLeuValSerLeuMetMetValGluLysValPro    130135140    AspSerThrTyrGluMetIleGlyGlyLeuAspLysGlnIleLysGlu    145150155160    IleLysGluValIleGluLeuProValLysHisProGluLeuPheGlu    165170175    AlaLeuGlyIleAlaGlnProLysGlyValLeuLeuTyrGlyProPro    180185190    GlyThrGlyLysThrLeuLeuAlaArgAlaValAlaHisHisThrAsp    195200205    CysThrPheIleArgValSerGlySerGluLeuValGlnLysPheIle    210215220    GlyGluGlyAlaArgMetValArgGluLeuPheValMetAlaArgGlu    225230235240    HisAlaProSerIleIlePheMetAspGluIleAspSerIleGlySer    245250255    SerArgLeuGluGlyGlySerGlyGlySerSerGluValGlnArgGln    260265270    MetLeuGluLeuLeuAsnGlnLeuAspGlyPheGluAlaThrLysAsn    275280285    IleLysValIleMetAlaThrAsnArgIleAspMetLeuAspSerAla    290295300    LeuLeuArgProGlyArgIleAspArgLysIleGluPheProProPro    305310315320    AsnGluGluAlaArgLeuAspIleLeuLysIleHisSerArgLysMet    325330335    AsnLeuThrArgGlyIleAsnLeuArgLysIleAlaGluLeuMetPro    340345350    GlyAlaSerGlyAlaGluValLysGlyValCysThrGluAlaGlyMet    355360365    TyrAlaLeuArgGluArgArgValHisValThrGlnGluAspPheGlu    370375380    MetAlaValAlaLysValMetGlnLysAspSerGluLysAsnMetSer    385390395400    IleLysLysLeuTrpLys    405    (2) INFORMATION FOR SEQ ID NO: 2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 405    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: Not Relevant    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:    MetThrAlaAlaValThrSerSerAsnIleValLeuGluThrHisGlu    151015    SerGlyIleLysProTyrPheGluGlnLysIleGlnGluThrGluLeu    202530    LysIleArgSerLysThrGluAsnGlyArgArgLeuGluAlaGlnArg    354045    AsnAlaLeuAsnAspLysValArgPheIleLysAspGluLeuArgLeu    505560    LeuGlnGluProGlySerTyrValGlyGluValIleLysIleValSer    65707580    AspLysLysValLeuValLysValGlnProGluGlyLysTyrIleVal    859095    AspValAlaLysAspIleAsnValLysAspLeuLysAlaSerGlnArg    100105110    ValCysLeuArgSerAspSerTyrMetLeuHisLysValLeuGluAsn    115120125    LysAlaAspProLeuValSerIleMetMetValGluLysValProAsp    130135140    SerThrTyrAspMetValGlyGlyLeuThrLysGlnIleLysGluIle    145150155160    LysGluValIleGluLeuProValLysHisProGluLeuPheGluSer    165170175    LeuGlyIleAlaGlnProLysGlyValIleLeuTyrGlyProProGly    180185190    ThrGlyLysThrLeuLeuAlaArgAlaValAlaHisHisThrAspCys    195200205    LysPheIleArgValSerGlyAlaGluLeuValGlnLysTyrIleGly    210215220    GluGlySerArgMetValArgGluLeuPheValMetAlaArgGluHis    225230235240    AlaProSerIleIlePheMetAspGluIleAspSerIleGlySerThr    245250255    ArgValGluGlySerGlyGlyGlyAspSerGluValGlnArgThrMet    260265270    LeuGluLeuLeuAsnGlnLeuAspGlyPheGluThrSerLysAsnIle    275280285    LysIleIleMetAlaThrAsnArgLeuAspIleLeuAspProAlaLeu    290295300    LeuArgProGlyArgIleAspArgLysIleGluPheProProProSer    305310315320    ValAlaAlaArgAlaGluIleLeuArgIleHisSerArgLysMetAsn    325330335    LeuThrArgGlyIleAsnLeuArgLysValAlaGluLysMetAsnGly    340345350    CysSerGlyAlaAspValLysGlyValCysThrGluAlaGlyMetTyr    355360365    AlaLeuArgGluArgArgIleHisValThrGlnGluAspPheGluLeu    370375380    AlaValGlyLysValMetAsnLysAsnGlnGluThrAlaIleSerVal    385390395400    AlaLysLeuPheLys    405    (2) INFORMATION FOR SEQ ID NO: 3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 185    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: Not Relevant    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:    MetProGlyProLeuArgGlyGlnHisPheTyrAlaValGluArgArg    151015    AlaTyrCysGluGlyCysTyrValAlaThrLeuGluLysCysAlaThr    202530    CysSerGlnProIleLeuAspArgIleLeuArgAlaMetGlyLysAla    354045    TyrHisProGlyCysPheThrCysValValCysHisArgGlyLeuAsp    505560    GlyIleProPheThrValAspAlaThrSerGlnIleHisCysIleGlu    65707580    AspPheHisArgLysPheAlaProArgCysSerValCysGlyGlyAla    859095    IleMetProGluProGlyGlnGluGluThrValArgIleValAlaLeu    100105110    AspArgSerPheHisIleGlyCysTyrLysCysGluGluCysGlyLeu    115120125    LeuLeuSerSerGluGlyGluCysGlnGlyCysTyrProLeuAspGly    130135140    HisIleLeuCysLysAlaCysArgProGlyAlaSerArgSerSerGln    145150155160    ProProSerGlyLeuThrAlaGluSerSerMetLysTyrLeuLeuGly    165170175    SerGlnPheGlnPheProSerPheAsp    180185    (2) INFORMATION FOR SEQ ID NO: 4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 122    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: Not Relevant    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:    CysAlaThrCysSerGlnProIleLeuAspArgIleLeuArgAlaMet    151015    GlyLysAlaTyrHisProGlyCysPheThrCysValValCysHisArg    202530    GlyLeuAspGlyIleProPheThrValAspAlaThrSerGlnIleHis    354045    CysIleGluAspPheHisArgLysPheAlaProArgCysSerValCys    505560    GlyGlyAlaIleMetProGluProGlyGlnGluGluThrValArgIle    65707580    ValAlaLeuAspArgSerPheHisIleGlyCysTyrLysCysGluGlu    859095    CysGlyLeuLeuLeuSerSerGluGlyGluCysGlnGlyCysTyrPro    100105110    LeuAspGlyHisIleLeuCysLysAlaCys    115120    (2) INFORMATION FOR SEQ ID NO: 5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 114    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: Not Relevant    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:    CysAlaAlaCysAlaGlnProIleLeuAspArgTyrValPheThrVal    151015    LeuGlyLysCysTrpHisGlnSerCysLeuArgCysCysAspCysArg    202530    AlaProMetSerMetThrCysPheSerArgAspGlyLeuIleLeuCys    354045    LysThrAspPheSerArgArgTyrSerGlnArgCysAlaGlyCysAsp    505560    GlyLysLeuGluLysGluAspLeuValArgArgAlaArgAspLysVal    65707580    PheHisIleArgCysPheGlnCysSerValCysGlnArgLeuLeuAsp    859095    ThrGlyAspGlnLeuTyrIleMetGluGlyAsnArgPheValCysGln    100105110    SerAsp    (2) INFORMATION FOR SEQ ID NO: 6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 495    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:    AACCCAATTCTTACCAGTTTGTTGCAAATCACAGGGAACNGGGGGTCT48    AsnProIleLeuThrSerLeuLeuGlnIleThrGlyAsnXaaGlySer    151015    ACCATTGGCTCGAGTCCGACCCCTCCTCATCACACGCCGCCACCTGTC96    ThrIleGlySerSerProThrProProHisHisThrProProProVal    202530    TCTTCGATGGCCGGCAACACCAAGAACCACCCGATGCTCATGAACCTT144    SerSerMetAlaGlyAsnThrLysAsnHisProMetLeuMetAsnLeu    354045    CTTAAAGATAATCCTGCCCAGGATTTCTCAACCCTTTATGGAAGCAGC192    LeuLysAspAsnProAlaGlnAspPheSerThrLeuTyrGlySerSer    505560    CCTTTAGAAAGGCAGAACTCCTCTTTCGGCTCACCCCGCATGGAAATA240    ProLeuGluArgGlnAsnSerSerPheGlySerProArgMetGluIle    65707580    TGCTCGGGGAGCAACAAGACCAAGAAAAAGAAGTCATCAAGATTACCA288    CysSerGlySerAsnLysThrLysLysLysLysSerSerArgLeuPro    859095    CCTGAGAAACCAAAACAACGCGAGGATATAATTGCCAAAACCAGGCTT336    ProGluLysProLysGlnArgGluAspIleIleAlaLysThrArgLeu    100105110    GAGGTTGGTGACTCTTGAAAGATTTTCTTTCTTCAGGCCTAGATCAGA384    GluValGlyAspSerLysIlePhePheLeuGlnAlaIleArg    115120125    AAATTAAGTGCAGCAATATCATGAATTCTCAGAAGCCCTTTCAGGGAG432    LysLeuSerAlaAlaIleSerIleLeuArgSerProPheArgGlu    130135140    CCAGTGAGTCATACAGTATCCACAGTTGAGTCACTTAAAGATGTCAGT480    ProValSerHisThrValSerThrValGluSerLeuLysAspValSer    145150155160    ATACGAAACATTATT495    IleArgAsnIleIle    165    (2) INFORMATION FOR SEQ ID NO: 7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 885    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:    CTCAAATGTAGCACCGTCGTCTGCGTGATCTGCTTGGAGAAGCCCAAA48    LeuLysCysSerThrValValCysValIleCysLeuGluLysProLys    151015    TACCGCTGTCCAGCCTGCCGCGTGCCCTACTGCTCGGTAGTCTGCTTC96    TyrArgCysProAlaCysArgValProTyrCysSerValValCysPhe    202530    CGGAAGCACAAAGAACAGTGCAACCCTGAAACTCGTCCTGTTGAGAAA144    ArgLysHisLysGluGlnCysAsnProGluThrArgProValGluLys    354045    AAAATAAGATCAGCTCTTCCTACCAAAACCGTAAAGCCTGTGGAAAAC192    LysIleArgSerAlaLeuProThrLysThrValLysProValGluAsn    505560    AAAGATGATGATGACTCTATAGCTGATTTTCTCAATAGTGATGAGGAA240    LysAspAspAspAspSerIleAlaAspPheLeuAsnSerAspGluGlu    65707580    GAAGACAGAGTTTCTTTGCAGAATTTAAAGAATTTAGGGGAATCTGCA288    GluAspArgValSerLeuGlnAsnLeuLysAsnLeuGlyGluSerAla    859095    ACATTAAGAAGCTTATTGCTCAATCCACACCTCAGGCAGTTGATGGTC336    ThrLeuArgSerLeuLeuLeuAsnProHisLeuArgGlnLeuMetVal    100105110    AACCTCGATCAGGGAGAAGACAAAGCAAAGCTCATGAGAGCTTACATG384    AsnLeuAspGlnGlyGluAspLysAlaLysLeuMetArgAlaTyrMet    115120125    CAAGAGCCTTTGTTTGTGGAGTTTGCAGACTGCTGTTTAGGAATTGTG432    GlnGluProLeuPheValGluPheAlaAspCysCysLeuGlyIleVal    130135140    GAGCCATCCCAGAATGAGGAGTCTTAAGATGGATTATTGTGCTGCTTG480    GluProSerGlnAsnGluGluSerAspGlyLeuLeuCysCysLeu    145150155160    CTCAAGCGTGTGCTTGACTCCTGGAACCTGCCTGCTCCCTCTCCCAGA528    LeuLysArgValLeuAspSerTrpAsnLeuProAlaProSerProArg    165170175    CCAGCTAGTTTGGGGCTGGGGAGCTCAGGCAAAAGAGGTTTCCAGGAT576    ProAlaSerLeuGlyLeuGlySerSerGlyLysArgGlyPheGlnAsp    180185190    GCAGATTAGGTCATGCAGGCCTTTACCGGCATTGATGTGGCTCATGTT624    AlaAspValMetGlnAlaPheThrGlyIleAspValAlaHisVal    195200205    TCAGGCAGACTTGGGGTCCTTAAGGTGGCAAGTCCTTTATGGAGAGAA672    SerGlyArgLeuGlyValLeuLysValAlaSerProLeuTrpArgGlu    210215220    AACTTGACATTCAGATGATTGTTTTTAAATGTTTTACTTTTGGTACAG720    AsnLeuThrPheArgLeuPheLeuAsnValLeuLeuLeuValGln    225230235240    TTGATAGACATCATAAACGATATCAAGCTTACACTTCATATGGAGTTA768    LeuIleAspIleIleAsnAspIleLysLeuThrLeuHisMetGluLeu    245250255    AACTTGGTCAGTGTTAATAAAATCAAAACGTGATTCTACTGTACATTG816    AsnLeuValSerValAsnLysIleLysThrPheTyrCysThrLeu    260265270    CATTATTCATAATTTAATTGTTTGAAATTACATTAAATAAATCAACTA864    HisTyrSerPheAsnCysLeuLysLeuHisIleAsnGlnLeu    275280285    ATTAAAAAAAAAAAAAAAAAA885    IleLysLysLysLysLysLys    290295    (2) INFORMATION FOR SEQ ID NO: 8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 201    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:    TCGCTCGTGCTCGCCCGCGCCTGGCCTACCGCGGCACTCCCGGCTGCA48    SerLeuValLeuAlaArgAlaTrpProThrAlaAlaLeuProAlaAla    151015    CGCTCTGCTTGGCCTCGCATGCCGGTGGACCTCAGCAAGTGGTCCGGG96    ArgSerAlaTrpProArgMetProValAspLeuSerLysTrpSerGly    202530    CCCTTGAGCCTGCAAGAAGTGGACGAGCAGCCGCAGCACCCGCTGCAT144    ProLeuSerLeuGlnGluValAspGluGlnProGlnHisProLeuHis    354045    GTCACCTACGCCGGGGCGCGTGGACGAGCTGGGCAACGTGCTGACGCC192    ValThrTyrAlaGlyAlaArgGlyArgAlaGlyGlnArgAlaAspAla    505560    CACCCAGGT201    HisProGly    65    (2) INFORMATION FOR SEQ ID NO: 9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 237    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:    TCTCAAGAGACTGAACAGAGATGTGAATCTCTGAACACAAGAACAGTT48    SerGlnGluThrGluGlnArgCysGluSerLeuAsnThrArgThrVal    151015    TATTTTTCTGAACAGTGGGTATCTTCCTTAAATGAAAGGGAACAGGAA96    TyrPheSerGluGlnTrpValSerSerLeuAsnGluArgGluGlnGlu    202530    CTTCACAACTTATTGGAGGTTGTAAGCCAATGTTGTGAGGCTTCAAGT144    LeuHisAsnLeuLeuGluValValSerGlnCysCysGluAlaSerSer    354045    TCAGACATCACTGAGAAATCAGATGGACGTAAGGCAGCTCATGAGAAA192    SerAspIleThrGluLysSerAspGlyArgLysAlaAlaHisGluLys    505560    CAGCATAACATTTTTCTTGATCAGATGACTATTGATGAAGATAAA237    GlnHisAsnIlePheLeuAspGlnMetThrIleAspGluAspLys    657075    (2) INFORMATION FOR SEQ ID NO: 10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 126    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:    GAAGATCAAGATACCTCAAAGAATTCTAAGCTAAACTCACACCAGAAA48    GluAspGlnAspThrSerLysAsnSerLysLeuAsnSerHisGlnLys    151015    GTAACACTTCTTCAATTGCTACTTGGCCATAAGAATGAAGAAAATGTA96    ValThrLeuLeuGlnLeuLeuLeuGlyHisLysAsnGluGluAsnVal    202530    GAAAAAAACACCAGCTGCAGGTGATGATGA126    GluLysAsnThrSerCysArg    3540    (2) INFORMATION FOR SEQ ID NO: 11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 570    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:    CTTACCTTAGAAAACCAAATTAAAGAAGAAAGAGAACAAGACAACTCT48    LeuThrLeuGluAsnGlnIleLysGluGluArgGluGlnAspAsnSer    151015    GAATCTCCAAATGGCAGAACATCACCTCTTGTGTCCCAGAATAATGAA96    GluSerProAsnGlyArgThrSerProLeuValSerGlnAsnAsnGlu    202530    CAAGGCTCAACCTTACGGGATTTGCTGACTACAACAGCTGGAAAGCTA144    GlnGlySerThrLeuArgAspLeuLeuThrThrThrAlaGlyLysLeu    354045    CGTGTGGGGTCTACAGATGCTGGCATTGCCTTTGCCCCAGTATATGCA192    ArgValGlySerThrAspAlaGlyIleAlaPheAlaProValTyrAla    505560    ATGGGAGCCCCAAGTAGCAAAAGTGGACGGACTATGCCTAACATTCTT240    MetGlyAlaProSerSerLysSerGlyArgThrMetProAsnIleLeu    65707580    GATGACATAATTGCTTCAGTTGTTGAAAACAAAATTCCACCAAGTAAA288    AspAspIleIleAlaSerValValGluAsnLysIleProProSerLys    859095    ACCTCCAAGATAAATGTAAAACCAGAGCTTAAAGAAGAGCCTGAAGAA336    ThrSerLysIleAsnValLysProGluLeuLysGluGluProGluGlu    100105110    AGCATAATATCTGCAGTGGATGAAAATAATAAATTATACAGTGATATA384    SerIleIleSerAlaValAspGluAsnAsnLysLeuTyrSerAspIle    115120125    CCACATTCTTGGATCTGTGAGAAGCATATTTTATGGCTTAGGATTATA432    ProHisSerTrpIleCysGluLysHisIleLeuTrpLeuArgIleIle    130135140    AGAATAGCAGTAATTGGAAGCTTTTCAAAGAATGTTGGAAACAAGGAC480    ArgIleAlaValIleGlySerPheSerLysAsnValGlyAsnLysAsp    145150155160    AGCCTGCAGTGGTTTCTGGTGTGCATAAGAAAATGAACATTAGCCTAT528    SerLeuGlnTrpPheLeuValCysIleArgLysThrLeuAlaTyr    165170175    GGAAGGCGGAATCAATTAGTCTTGATTTTGGAGACCACCAAG570    GlyArgArgAsnGlnLeuValLeuIleLeuGluThrThrLys    180185190    (2) INFORMATION FOR SEQ ID NO: 12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 624    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:    AACCATACCCCTGGCGCCTTGTACCCCGATTCCGACTTGGAGAAGGAA48    AsnHisThrProGlyAlaLeuTyrProAspSerAspLeuGluLysGlu    151015    GAAGAGGAGAGTGAGGAGGACTGGAAGCTGCAGCTGGAGGCTGAAAAC96    GluGluGluSerGluGluAspTrpLysLeuGlnLeuGluAlaGluAsn    202530    TACGAGGGCCACACCCCACTCCACGTGGCCGTTATCCACAAAGATGTG144    TyrGluGlyHisThrProLeuHisValAlaValIleHisLysAspVal    354045    GAGATGGTCCGGCTGCTCCGAGATGCTGGAGCTGACCTTGACAAACCG192    GluMetValArgLeuLeuArgAspAlaGlyAlaAspLeuAspLysPro    505560    GAGCCCACGTGCGGCCGGAGCCCCTTCATTTGGCAGTGGAGGCCAGGC240    GluProThrCysGlyArgSerProPheIleTrpGlnTrpArgProGly    65707580    AGCCGATGTGCTGGAGCTTCTCTGAGGGCAGGCGCGAACCCTGCTGCC288    SerArgCysAlaGlyAlaSerLeuArgAlaGlyAlaAsnProAlaAla    859095    CGCATGTACGGTGGCCGCACCCCACTCGGCAGTGCCATGCTCCGGCCC336    ArgMetTyrGlyGlyArgThrProLeuGlySerAlaMetLeuArgPro    100105110    AACCCCATCCTCGCCCGCCTCCTCCGTGCACACGGAGCCCCTGAGCCC384    AsnProIleLeuAlaArgLeuLeuArgAlaHisGlyAlaProGluPro    115120125    GAGGGGAAGGACGAGAAATCCGGCCCCTGCAGCAGCAGTAGCGAGCAC432    GluGlyLysAspGluLysSerGlyProCysSerSerSerSerGluHis    130135140    GACNAGAGANGACGAGGGCGATGAATACGACGACATTGTGGTTCACAG480    AspXaaArgXaaArgGlyArgIleArgArgHisCysGlySerGln    145150155160    CAGCCGCAGCCAAACCCGGCTGCCTCCCACCCCAGCCTCAAAACCTCT528    GlnProGlnProAsnProAlaAlaSerHisProSerLeuLysThrSer    165170175    TCCTGACGACCCCCGCCCCGTGTGATTTGTTTCATTGTTAATATAATT576    SerArgProProProArgValIleCysPheIleValAsnIleIle    180185190    TCCAGTTTAATAAACAAAACCCTAGTTCTGACAACCAGAAAAAAAAAA624    SerSerLeuIleAsnLysThrLeuValLeuThrThrArgLysLysLys    195200205    (2) INFORMATION FOR SEQ ID NO: 13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 99    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:    AGACACCCGCTGATCAGAGACATGCTTCGACGAATTAAGGAAGAAGAG48    ArgHisProLeuIleArgAspMetLeuArgArgIleLysGluGluGlu    151015    GATCTGGGTAAAAGTAGAGAAGGATCAAGGACGGATGATGAAGTAGTA96    AspLeuGlyLysSerArgGluGlySerArgThrAspAspGluValVal    202530    CAG99    Gln    (2) INFORMATION FOR SEQ ID NO: 14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 216    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:    CAGGTGGAAGAAAACACCCCGTACTGGCAGGCATGGAGCCAACAAGGA48    GlnValGluGluAsnThrProTyrTrpGlnAlaTrpSerGlnGlnGly    151015    GAACCTGGAGCTCAACGGCAGCATCCTGAGTGCGAGAACTTTCAAAGG96    GluProGlyAlaGlnArgGlnHisProGluCysGluAsnPheGlnArg    202530    CTTCCAAATCTGATGCTACTTCTGGAATCCTCAATTCAACCAACATCC144    LeuProAsnLeuMetLeuLeuLeuGluSerSerIleGlnProThrSer    354045    AGTCCTGAGAAGCCCTGATCAGTCAACCAGCTGTGGCTTCCTGTGCCT192    SerProGluLysProSerValAsnGlnLeuTrpLeuProValPro    505560    AGACTGGACCTAATTATATGGGGG216    ArgLeuAspLeuIleIleTrpGly    6570    (2) INFORMATION FOR SEQ ID NO: 15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 634    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:    TGCCGCTGCAGCAGCGCAGTTCCAGTCCGTTGCTTTACTTTTTGCTTC48    CysArgCysSerSerAlaValProValArgCysPheThrPheCysPhe    151015    ACCGACATAGTCATTATGCCGAAGAGAAAGTCTCCAGAGAATACAGAG96    ThrAspIleValIleMetProLysArgLysSerProGluAsnThrGlu    202530    GGCAAAGATGGATCCAAAGTAACTAAACAGGAGCCCACAAGACGGTCT144    GlyLysAspGlySerLysValThrLysGlnGluProThrArgArgSer    354045    GCCAGATTGTCAGCGAAACCTGCTCCACCAAAACCTGAACCCAAACCA192    AlaArgLeuSerAlaLysProAlaProProLysProGluProLysPro    505560    AGAAAAACATCTGCTAAGAAAGAACCTGGAGCAAAGATTAGCAGAGGT240    ArgLysThrSerAlaLysLysGluProGlyAlaLysIleSerArgGly    65707580    GCTAAAGGGAGGAAGGAGGAAAAGCAGGAAGCTGGAAAGGAAGGTACT288    AlaLysGlyArgLysGluGluLysGlnGluAlaGlyLysGluGlyThr    859095    GCACCATCTGAAAATGGTGAAACTAAAGCTGAAGAGGCACAGAAAACT336    AlaProSerGluAsnGlyGluThrLysAlaGluGluAlaGlnLysThr    100105110    GAATCTGTAGATAACGAGGGAGAATGAATTGTCATGAAAAATTGGGGT384    GluSerValAspAsnGluGlyGluIleValMetLysAsnTrpGly    115120125    TGATTTTATGTATCTCTTGGGACAACTTTTAAAAGCTATTTTTACCAA432    PheTyrValSerLeuGlyThrThrPheLysSerTyrPheTyrGln    130135140    GTATTTTGTAAATGCTAATTTTTTAGGACTCTACTAGTTGGCATACGA480    ValPheCysLysCysPhePheArgThrLeuLeuValGlyIleArg    145150155160    AAATATATAAGGATGGACATTTATCGTCTCATAGTCATGCTTTTTGGA528    LysTyrIleArgMetAspIleTyrArgLeuIleValMetLeuPheGly    165170175    ATTTNNNNNNNNNNNNNNNNNNNNNNNNNNNCAGGAAGTTTGCCCCAA576    IleXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaGlySerLeuProGln    180185190    GATGCTCAGTGTGCCGTGGGGCCATAACTGCCTGAGCCAGGTCAGGAG624    AspAlaGlnCysAlaValGlyProLeuProGluProGlyGlnGlu    195200205    GAGACTGCTG634    GluThrAla    210    (2) INFORMATION FOR SEQ ID NO: 16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 638    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:    AAACATCCTATCATCTGTAGGCTCATTCATTTCTCTAACAGCAGCAGC48    LysHisProIleIleCysArgLeuIleHisPheSerAsnSerSerSer    151015    AACAGCGCATCACAGGACACCAAGGAGAGCTCTGAAGAGCCTCCCTCA96    AsnSerAlaSerGlnAspThrLysGluSerSerGluGluProProSer    202530    GAAGAGAGCCAGGACACCCCCATTTACACGGAGTTTGATGAGGATTTC144    GluGluSerGlnAspThrProIleTyrThrGluPheAspGluAspPhe    354045    GAGGAGGAACCCACATCCCCCATAGGTCACTGTGTGGCCATCTACCAC192    GluGluGluProThrSerProIleGlyHisCysValAlaIleTyrHis    505560    TTTGAAGGGTCCAGCGAGGGCACTATCTCTATGGCCGAGGGTGAAGAC240    PheGluGlySerSerGluGlyThrIleSerMetAlaGluGlyGluAsp    65707580    CTCAGTCTTATGGAAGAAGACAAAGGGGACGGCTGGACCCGGGTCAGG288    LeuSerLeuMetGluGluAspLysGlyAspGlyTrpThrArgValArg    859095    CGGAAAGAGGGAGGCGAGGGCTACGTGCCCACCTCCTACCTCCGAGTC336    ArgLysGluGlyGlyGluGlyTyrValProThrSerTyrLeuArgVal    100105110    ACGCTCAATTGAACCCTGCCAGAGACGGGAAGAGGGGGGCTGTCGGCT384    ThrLeuAsnThrLeuProGluThrGlyArgGlyGlyLeuSerAla    115120125    GCTGCTTCTGGGCCACGGGGAGCCCCAGGACCTATGCACTTTATTTCT432    AlaAlaSerGlyProArgGlyAlaProGlyProMetHisPheIleSer    130135140    GACCCCGTGGCTTCGGCTGAGACCTGTGTAACCTGCTGCCCCCTCCAC480    AspProValAlaSerAlaGluThrCysValThrCysCysProLeuHis    145150155160    CCCCAACCCAGTCCTACCTGTCACACCGGACGGACCCGCTGTGCCTTC528    ProGlnProSerProThrCysHisThrGlyArgThrArgCysAlaPhe    165170175    TACCATCGTTCCACCATTGATGTACATACTCATGTTTTACATCTTTTC576    TyrHisArgSerThrIleAspValHisThrHisValLeuHisLeuPhe    180185190    TTTCTGCGCTCGGCTCCGGCCATTTTGTTTTATACAAAAATGGGAAAA624    PheLeuArgSerAlaProAlaIleLeuPheTyrThrLysMetGlyLys    195200205    AAAAAAAAAAAAAA638    LysLysLysLys    210    (2) INFORMATION FOR SEQ ID NO: 17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 862    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:    GGCACGAGGCGTGACGTCCGACAAGAAATGCTGGATGATGTACAAAAG48    GlyThrArgArgAspValArgGlnGluMetLeuAspAspValGlnLys    151015    AAATTGATGAGCTTAGCAAACAGCTCAGAAGGAAAAGTAGACAAAGTC96    LysLeuMetSerLeuAlaAsnSerSerGluGlyLysValAspLysVal    202530    CTAATGAGAAACCTCTTCATTGGTCATTTCCACACACCGAAAAATCAG144    LeuMetArgAsnLeuPheIleGlyHisPheHisThrProLysAsnGln    354045    CGTCATGAAGTGTTACGGTTAATGGGGAGCATCCTGGGCGTCAGAAGG192    ArgHisGluValLeuArgLeuMetGlySerIleLeuGlyValArgArg    505560    GAGGAGATGGAGCAGTTGTTTCATGACGATCAGGGCAGTGTTACCAGG240    GluGluMetGluGlnLeuPheHisAspAspGlnGlySerValThrArg    65707580    TGGATGACTGGGTGGCTTGGAGGAGGATCAAAAAGTGTTCCCAACACA288    TrpMetThrGlyTrpLeuGlyGlyGlySerLysSerValProAsnThr    859095    CCTTTGAGACCAAATCAGCAATCTGTGGTTAATAGTTCTTTTTCAGAA336    ProLeuArgProAsnGlnGlnSerValValAsnSerSerPheSerGlu    100105110    CTTTTTGTTAAATTTCTAGAAACAGAATCTCATCCATCCATTCCACCA384    LeuPheValLysPheLeuGluThrGluSerHisProSerIleProPro    115120125    CCAAAGCTTTCTGTTCATGATATGAAACCTCTGGATTCACCAGGAAGA432    ProLysLeuSerValHisAspMetLysProLeuAspSerProGlyArg    130135140    AGAAAAAGAGATACAAATGCACCAGAAAGTTTTAAAGATACAGCAGAA480    ArgLysArgAspThrAsnAlaProGluSerPheLysAspThrAlaGlu    145150155160    TCCAGGTCTGGTAGAAGAACAGATGTAAATCCGTTTTTGGCTCCTCGC528    SerArgSerGlyArgArgThrAspValAsnProPheLeuAlaProArg    165170175    TCGGCAGCTGTACCTCTTATTAACCCAGCTGGACTTGGACCTGGTGGG576    SerAlaAlaValProLeuIleAsnProAlaGlyLeuGlyProGlyGly    180185190    CCGGGCATCTTCTTCTGAAACCCATCTCAGATGTTTTGCCCACATTTA624    ProGlyIlePhePheAsnProSerGlnMetPheCysProHisLeu    195200205    CACCTTTGCCAGCGTTACCTGACAACAGTGCTGGGGTTGTGCTGAAAG672    HisLeuCysGlnArgTyrLeuThrThrValLeuGlyLeuCysLys    210215220    CCTTTTAAAGCAATAGATGATTCTCAAGCCAGAGACAATCTAGCACTT720    ProPheLysAlaIleAspAspSerGlnAlaArgAspAsnLeuAlaLeu    225230235240    TAAAGAAACCATGAACACTATATGTATGTACTTTATCACAAAGTGGCC768    ArgAsnHisGluHisTyrMetTyrValLeuTyrHisLysValAla    245250255    TTTGGGGAGAAAGTCATGTATTTGTTCGCAATTATGCTTTCTCTGAAT816    PheGlyGluLysValMetTyrLeuPheAlaIleMetLeuSerLeuAsn    260265270    TTAATAAAAATATTCCTAATGCTTTTAGAAAAAAAAAAAAAAAAAA862    LeuIleLysIlePheLeuMetLeuLeuGluLysLysLysLysLys    275280285    (2) INFORMATION FOR SEQ ID NO: 18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 247    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:    GGCACGAGGCGAGTTCTCCCACCTGAGCAGAAATATGACCATGCAGCG48    GlyThrArgArgValLeuProProGluGlnLysTyrAspHisAlaAla    151015    CACCATGAAGCTCTACCGACTGCCAGAGACTCCCAAGACAGCTGGGCT96    HisHisGluAlaLeuProThrAlaArgAspSerGlnAspSerTrpAla    202530    GCGACCAATGGAAACAAAGGACATTCCAGTAGTGCACCAGCTCCTCAC144    AlaThrAsnGlyAsnLysGlyHisSerSerSerAlaProAlaProHis    354045    CAGGTACTTGAAGCAATTTCACCTTACGCCCGTCATGAGCCAGGAGGA192    GlnValLeuGluAlaIleSerProTyrAlaArgHisGluProGlyGly    505560    GGTGGAGCACTGGTTCTACCCCCAGGAGAATATCATCGACACTTTCGT240    GlyGlyAlaLeuValLeuProProGlyGluTyrHisArgHisPheArg    65707580    GGTGGAG247    GlyGly    (2) INFORMATION FOR SEQ ID NO: 19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 102    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:    AGGGCGCACCTGGAGCTGTTCTGGTCTAGAGTGAATATCCCCAAGGTG48    ArgAlaHisLeuGluLeuPheTrpSerArgValAsnIleProLysVal    151015    CTAAGAGCTGCAGAACAAGCTCATCTTTGGGCAGACTGGTGTTTTTGT96    LeuArgAlaAlaGluGlnAlaHisLeuTrpAlaAspTrpCysPheCys    202530    ATGACA102    MetThr    (2) INFORMATION FOR SEQ ID NO: 20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 219    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:    GTTAGCTCTAGAGGCCATTCTTTTGCTGATCCTGCCAGTAATCTTGGG48    ValSerSerArgGlyHisSerPheAlaAspProAlaSerAsnLeuGly    151015    CTGGAAGACATTATCAGGAAGGCTCTCATGGGAAGCTTTGATGACAAA96    LeuGluAspIleIleArgLysAlaLeuMetGlySerPheAspAspLys    202530    GTTGAGGATCATGGAGTTGTCATGTCCCAGCCTATGGGAGTAGTGCCT144    ValGluAspHisGlyValValMetSerGlnProMetGlyValValPro    354045    GGTACTGCCAACACCGATTGCATGTGCTCCCTCTGCGGTGAACCAAGC192    GlyThrAlaAsnThrAspCysMetCysSerLeuCysGlyGluProSer    505560    AGCTCCTCACCAACAGAACAGGATCTG219    SerSerSerProThrGluGlnAspLeu    6570    (2) INFORMATION FOR SEQ ID NO: 21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 553    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:    AATATCGAACTGAAGAAAGGAGGGAAGGATATACCAGTCACTATCCAC48    AsnIleGluLeuLysLysGlyGlyLysAspIleProValThrIleHis    151015    AATTTAGAGGAGTATCTAAGACTGGTTATATTCTGGGCACTAAATGAA96    AsnLeuGluGluTyrLeuArgLeuValIlePheTrpAlaLeuAsnGlu    202530    GGCGTTTCTAGGCAATTTGATTCGTTCAGAGATGGATTTGAATCAGTC144    GlyValSerArgGlnPheAspSerPheArgAspGlyPheGluSerVal    354045    TTCCCACTCAGTCATCTTCAGTACTTCTACCCGGAGGAACTGGATCAG192    PheProLeuSerHisLeuGlnTyrPheTyrProGluGluLeuAspGln    505560    CTCCTTTGTGGCAGTAAAGCAGACACTTGGGATGCAAAGACACTGATG240    LeuLeuCysGlySerLysAlaAspThrTrpAspAlaLysThrLeuMet    65707580    GAATGCTGTAGGCCTGATCATGGTTATACTCATGACAGTCGGGCTGTG288    GluCysCysArgProAspHisGlyTyrThrHisAspSerArgAlaVal    859095    AAGTTTTTGTTTGAGATTCTCAGTAGTTTTGATAATGAGCAGCAGAGG336    LysPheLeuPheGluIleLeuSerSerPheAspAsnGluGlnGlnArg    100105110    TTATTTCTCCAGTTTGTGACTGGTAGCCCAAGATTGCCTGTTGGAGGA384    LeuPheLeuGlnPheValThrGlySerProArgLeuProValGlyGly    115120125    TTCCGGAGTTTGAATCCACCTTTGACAATTGTCCGAAAGACGTTTGAA432    PheArgSerLeuAsnProProLeuThrIleValArgLysThrPheGlu    130135140    TCAACAGAAAACCCAGATGACTTCTTGCCCTCTGTAATGACTTGTGTG480    SerThrGluAsnProAspAspPheLeuProSerValMetThrCysVal    145150155160    AACTATCTTAAGTTGCCGGACTATCAAGCATTGAGATATGCGTGAAAA528    AsnTyrLeuLysLeuProAspTyrGlnAlaLeuArgTyrAlaLys    165170175    ACTGTTGATAGCAGCAAGAGAAGGG553    ThrValAspSerSerLysArgArg    180    (2) INFORMATION FOR SEQ ID NO: 22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 186    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:    GAAGCAAAAAACGAGCCCTGGAAGAAGAAAAACCACGCCGGGAAATCC48    GluAlaLysAsnGluProTrpLysLysLysAsnHisAlaGlyLysSer    151015    TGGAAAAACGATTACAGGAAGAAACTAGCCAGAGGAGAAGTTAATAGA96    TrpLysAsnAspTyrArgLysLysLeuAlaArgGlyGluValAsnArg    202530    AAAGGAAGTAAAAATAAGGGAGAGACAAAGGGCACAGGCTCGTCCTTT144    LysGlySerLysAsnLysGlyGluThrLysGlyThrGlySerSerPhe    354045    GACACGCTACCTGCCTGTCCGGAAGAAGACTTTGATTTGCGG186    AspThrLeuProAlaCysProGluGluAspPheAspLeuArg    505560    (2) INFORMATION FOR SEQ ID NO: 23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 66    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:    AGGGTACGGGAAGCTGCTGAAAAGGCTAAGTCTGAACTCTCCTCATCT48    ArgValArgGluAlaAlaGluLysAlaLysSerGluLeuSerSerSer    151015    GTGCAGACTGACATCAAT66    ValGlnThrAspIleAsn    20    (2) INFORMATION FOR SEQ ID NO: 24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 192    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:    CATTTGAATATGAAGTTGACCCGTGCTCAATTTGAAGGGATTGTCACT48    HisLeuAsnMetLysLeuThrArgAlaGlnPheGluGlyIleValThr    151015    GATCTAATCAGAAGGACTATCGCTCCATGCCAAAAAGCTATGCAAGAT96    AspLeuIleArgArgThrIleAlaProCysGlnLysAlaMetGlnAsp    202530    GCAGAAGTCAGCAAGAGTGACATAGGAGAAGTGATTCTTGTGGGTGGC144    AlaGluValSerLysSerAspIleGlyGluValIleLeuValGlyGly    354045    ATGACTAGGATGCCCAAGGTTCAGCAGACTGTACAGGACTTTTTGGCA192    MetThrArgMetProLysValGlnGlnThrValGlnAspPheLeuAla    505560    (2) INFORMATION FOR SEQ ID NO: 25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 582    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:    GGGGGCAGTGGACGAGGCCGTGGCGACCTGAAGCAGGCGCTTCCCTGT48    GlyGlySerGlyArgGlyArgGlyAspLeuLysGlnAlaLeuProCys    151015    GTGGCCGAGTCGCCAACGGTCCACGTGGAGGTGCATCAGCGCGGCAGC96    ValAlaGluSerProThrValHisValGluValHisGlnArgGlySer    202530    AGCACTGCAAAGAAAGAAGACATAAACCTGAGTGTTAGAAAGCTACTC144    SerThrAlaLysLysGluAspIleAsnLeuSerValArgLysLeuLeu    354045    AACAGACATAATATTGTGTTTGGCGATTACACATGGACTGAGTTTGAT192    AsnArgHisAsnIleValPheGlyAspTyrThrTrpThrGluPheAsp    505560    GAACCTTTTTTGACCAGAAATGTGCAGTCTGTGTCTATTATTGACACA240    GluProPheLeuThrArgAsnValGlnSerValSerIleIleAspThr    65707580    GAATTAAAGGTTAAAGACTCACAGCCCATCGATTTGAGTGCATGCACT288    GluLeuLysValLysAspSerGlnProIleAspLeuSerAlaCysThr    859095    GTTGCACTTCACATTTTCCAGCTGAATGAAGATGGCCCCAGCAGTGAA336    ValAlaLeuHisIlePheGlnLeuAsnGluAspGlyProSerSerGlu    100105110    AATCTGGAGGAAGAGACAGAAAACATAATTGCAGCAAATCACTGGGTT384    AsnLeuGluGluGluThrGluAsnIleIleAlaAlaAsnHisTrpVal    115120125    CTACCTGCAGCTGAATTCCATGGGCTTTGGGACAGCTTGGTATACGAT432    LeuProAlaAlaGluPheHisGlyLeuTrpAspSerLeuValTyrAsp    130135140    GTGGAAGTCAAATCCCATCTCCTCGATTATGTGATGACAACTTTACTG480    ValGluValLysSerHisLeuLeuAspTyrValMetThrThrLeuLeu    145150155160    TTTTCAGACAAGAACGTCAACAGCAACCTCATCACCATAGAGGGGTTC528    PheSerAspLysAsnValAsnSerAsnLeuIleThrIleGluGlyPhe    165170175    CTCCAGGCCCTGTCTCTGGCAGTGGACAAGCAGTTTGAAGAGAGAAAG576    LeuGlnAlaLeuSerLeuAlaValAspLysGlnPheGluGluArgLys    180185190    AAGCTT582    LysLeu    (2) INFORMATION FOR SEQ ID NO: 26:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 487    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:    TTCACCACTGTGATGGACCTGCTCCTGGAGTATGAAGTCATCTGTATC48    PheThrThrValMetAspLeuLeuLeuGluTyrGluValIleCysIle    151015    TACTGGACCAAGTACTACACACTCCACAATGCAATCATTGAGGATTGT96    TyrTrpThrLysTyrTyrThrLeuHisAsnAlaIleIleGluAspCys    202530    GTCAGAAAACAGCTCAAAAAAGAGAGGCCCATCATCCTGGATCCGGCC144    ValArgLysGlnLeuLysLysGluArgProIleIleLeuAspProAla    354045    GACCCCACCCTCAACGTGGCAGAAGGGTACAGATGGGACATCGTTGCT192    AspProThrLeuAsnValAlaGluGlyTyrArgTrpAspIleValAla    505560    CAGAGGGCCTCCCAGTGCCTGAAACAGGACTGTTGCTATGACAACAGG240    GlnArgAlaSerGlnCysLeuLysGlnAspCysCysTyrAspAsnArg    65707580    GAGAAGGGGATCTCCAGCTGGAACGTGAAGAGGGCACGAGACATCCAC288    GluLysGlyIleSerSerTrpAsnValLysArgAlaArgAspIleHis    859095    TTGACAGTGGAGCAGAGGGGTTACCCAGATTTCAACCTCATCGTGAAC336    LeuThrValGluGlnArgGlyTyrProAspPheAsnLeuIleValAsn    100105110    CCTTATGAGCCCATAAGGAAGGTTAAAGAGAAAATCCGGAGACCAGGG384    ProTyrGluProIleArgLysValLysGluLysIleArgArgProGly    115120125    GCTACTCTGGCCTGCAGCGTCTGTCCTTCCAGGTTCCTGGCAGTGAGA432    AlaThrLeuAlaCysSerValCysProSerArgPheLeuAlaValArg    130135140    GGCAGCTTCTCAGCAGCAGGTGCTCCTTAGCCAAATATGGGATCTTCT480    GlySerPheSerAlaAlaGlyAlaProProAsnMetGlySerSer    145150155160    CCCACAC487    ProThr    (2) INFORMATION FOR SEQ ID NO: 27:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 768    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:    ATGGAGGATGATTTCATGTGCGATGATGAGGAGGACTACGACCTGGAA48    MetGluAspAspPheMetCysAspAspGluGluAspTyrAspLeuGlu    151015    TACTCTGAAGATAGTAACTCCGAGCCAAATGTGGATTTGGAAAATCAG96    TyrSerGluAspSerAsnSerGluProAsnValAspLeuGluAsnGln    202530    TACTATAATTCCAAAGCATTAAAAGAAGATGACCCAAAAGCGGCATTA144    TyrTyrAsnSerLysAlaLeuLysGluAspAspProLysAlaAlaLeu    354045    AGCAGTTTCCAAAAGGTTTTGGAACTTGAAGGTGAAAAAGGAGAATGG192    SerSerPheGlnLysValLeuGluLeuGluGlyGluLysGlyGluTrp    505560    GGATTTAAAGCACTGAAACAAATGATTAAGATTAACTTCAAGTTGACA240    GlyPheLysAlaLeuLysGlnMetIleLysIleAsnPheLysLeuThr    65707580    AACTTTCCAGAAATGATGAATAGATATAAGCAGCTATTGACCTATATT288    AsnPheProGluMetMetAsnArgTyrLysGlnLeuLeuThrTyrIle    859095    CGGAGTGCAGTCACAAGAAATTATTCTGAAAAATCCATTAATTCTATT336    ArgSerAlaValThrArgAsnTyrSerGluLysSerIleAsnSerIle    100105110    CTTGATTATATCTCTACTTCTAAACAGATGGATTTACTGCAGGAATTC384    LeuAspTyrIleSerThrSerLysGlnMetAspLeuLeuGlnGluPhe    115120125    TATGAAACAACACTGGAAGCTTTGAAAGATGCTAAGAATGATAGACTG432    TyrGluThrThrLeuGluAlaLeuLysAspAlaLysAsnAspArgLeu    130135140    TGGTTTAAGACAAACACAAAGCTTGGAAAATTATATTTAGAACGAGAG480    TrpPheLysThrAsnThrLysLeuGlyLysLeuTyrLeuGluArgGlu    145150155160    GAATATGGAAAGCTTCAAAAAATTTTACGCCAGTTACATCAGTCGTGC528    GluTyrGlyLysLeuGlnLysIleLeuArgGlnLeuHisGlnSerCys    165170175    CAGACTGATGATGGAGAAGATGATCTGAAAAAAGGTACACAGTTATTA576    GlnThrAspAspGlyGluAspAspLeuLysLysGlyThrGlnLeuLeu    180185190    GAAATATATGCTTTGGAAATTCAAATGTACACAGCACAGAAAAATAAC624    GluIleTyrAlaLeuGluIleGlnMetTyrThrAlaGlnLysAsnAsn    195200205    AAAAAACTTAAAGCACTCTATGAACAGTCACTTCACATCAAGTCTGCC672    LysLysLeuLysAlaLeuTyrGluGlnSerLeuHisIleLysSerAla    210215220    ATCCCTCATCCACTGATTATGGGAGTTATCAGAGAATGTGGTGGTAAA720    IleProHisProLeuIleMetGlyValIleArgGluCysGlyGlyLys    225230235240    ATTGCACTTGGGGGAGGTGAATTTGAAAAGGCACACACTGATTTTTTT768    IleAlaLeuGlyGlyGlyGluPheGluLysAlaHisThrAspPhePhe    245250255    (2) INFORMATION FOR SEQ ID NO: 28:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1121    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:    GCAGAGGTTAAAACACCTTTTGATTTGGCCAAGGCACAAGAGAACAGC48    AlaGluValLysThrProPheAspLeuAlaLysAlaGlnGluAsnSer    151015    AACTCCGTAAAGAAGAAGACAAAGTTTGTCAATTTATACACAAGAGAA96    AsnSerValLysLysLysThrLysPheValAsnLeuTyrThrArgGlu    202530    AGACAGGACAGGCTTGCAGTCCTGCTCCCTGGTCGTCACCCTTGTGAT144    ArgGlnAspArgLeuAlaValLeuLeuProGlyArgHisProCysAsp    354045    TGCCTGGGCCAGAAGCACAAGCTCATCAATAACTGTCTGATCTGTGGG192    CysLeuGlyGlnLysHisLysLeuIleAsnAsnCysLeuIleCysGly    505560    CGCATTGTCTGTGAACAAGAAGGCTCAGGCCCTTGCTTATTCTGTGGC240    ArgIleValCysGluGlnGluGlySerGlyProCysLeuPheCysGly    65707580    ACTCTGGTGTGTACTCATGAGGAACAAGATATTTTACAGCGTGACTCA288    ThrLeuValCysThrHisGluGluGlnAspIleLeuGlnArgAspSer    859095    AACAAGAGCCAGAAACTGCTAAAGAAACTCATGTCAGGAGTGGAGAAT336    AsnLysSerGlnLysLeuLeuLysLysLeuMetSerGlyValGluAsn    100105110    TCTGGAAAGGTGGACATCTCTACCAAGGACCTTCTTCCTCATCAAGAA384    SerGlyLysValAspIleSerThrLysAspLeuLeuProHisGlnGlu    115120125    TTGCGAATTAAGTCTGGTCTGGAGAAGGCTATCAAGCATAAAGACAAA432    LeuArgIleLysSerGlyLeuGluLysAlaIleLysHisLysAspLys    130135140    CTGTTAGAGTTTGACAGAACTAGTATTCGAAGGACCCAAGTCATTGAT480    LeuLeuGluPheAspArgThrSerIleArgArgThrGlnValIleAsp    145150155160    GATGAGTCAGATTACTTTGCCAGTGATTCTAACCAATGGTTGTCCAAA528    AspGluSerAspTyrPheAlaSerAspSerAsnGlnTrpLeuSerLys    165170175    CTTGAGCGGGAAACCTTGCAGAAGCGAGAGGAGGAGCTGAGAGAACTT576    LeuGluArgGluThrLeuGlnLysArgGluGluGluLeuArgGluLeu    180185190    CGACACGCCTCTCGACTTTCTAAGAAGGTCACCATTGACTTTGCAGGA624    ArgHisAlaSerArgLeuSerLysLysValThrIleAspPheAlaGly    195200205    AGGAAGATCCTGGAAGAAGAAAATTCACTAGCAGAGTATCATAGCAGA672    ArgLysIleLeuGluGluGluAsnSerLeuAlaGluTyrHisSerArg    210215220    CTAGATGAGACAATACAGGCCATTGCCAATGGAACCTTGAACCAGCCA720    LeuAspGluThrIleGlnAlaIleAlaAsnGlyThrLeuAsnGlnPro    225230235240    CTGACCAAATTGGATAGATCTTCTGAAGAGCCTTTGGGAGTTCTGGTA768    LeuThrLysLeuAspArgSerSerGluGluProLeuGlyValLeuVal    245250255    AATCCCAACATGTACCAGTCCCCTCCCCAGTGGTTGACCACACAGGTG816    AsnProAsnMetTyrGlnSerProProGlnTrpLeuThrThrGlnVal    260265270    CAGCCTCACAGAAGAAGGCTTTCCGTTCTTCAGGATTTGGACTAGAGT864    GlnProHisArgArgArgLeuSerValLeuGlnAspLeuAspSer    275280285    TCAACTCATTTCAGCACCAGTTGCGAATCCAGGATCAAGAATTTCAGG912    SerThrHisPheSerThrSerCysGluSerArgIleLysAsnPheArg    290295300    AAGGCTTTGATGGTGGCTGGTGCCTCTCTGTACATCAGCCCTGGGTTC960    LysAlaLeuMetValAlaGlyAlaSerLeuTyrIleSerProGlyPhe    305310315320    TCTGCTTGTCAGAGGGATTAAAAGGGTGGAGGGCAGATCCTGGTACAC1008    SerAlaCysGlnArgAspLysGlyGlyGlyGlnIleLeuValHis    325330335    CCCCCACAGAGGACGACTTTGGATAGCAGCCACAGCTAAAAAATCCCT1056    ProProGlnArgThrThrLeuAspSerSerHisSerLysIlePro    340345350    CCCCTCAAGAAGTCTCAGAACTCCAGGCTACATATCGTCTTCTTCGTT1104    ProLeuLysLysSerGlnAsnSerArgLeuHisIleValPhePheVal    355360365    GGGAAGATGTGGAATTT1121    GlyLysMetTrpAsn    370    (2) INFORMATION FOR SEQ ID NO: 29:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 108    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:    GAAAGGGCCCTGACAGCACACACACTTAAACACAGTTTTCTGATAACT48    GluArgAlaLeuThrAlaHisThrLeuLysHisSerPheLeuIleThr    151015    TTGGAATTCACACCGTTGGACTAGTTAAAAACTTCTAAAATAATTTTT96    LeuGluPheThrProLeuAspLeuLysThrSerLysIleIlePhe    202530    TAAAATCTAATA108    AsnLeuIle    35    (2) INFORMATION FOR SEQ ID NO: 30:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 219    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:    CCAGGAACTGAGATCTTTAATCTGCCAGCAGTTACTACGTCAGGCTCA48    ProGlyThrGluIlePheAsnLeuProAlaValThrThrSerGlySer    151015    GTTAGCTCTAGAGGCCATTCTTTTGCTGATCCTGCCAGTAATCTTGGG96    ValSerSerArgGlyHisSerPheAlaAspProAlaSerAsnLeuGly    202530    CTGGAAGACATTATCAGGAAGGCTCTCATGGGAAGCTTTGATGACAAA144    LeuGluAspIleIleArgLysAlaLeuMetGlySerPheAspAspLys    354045    GTTGAGGATCATGGAGTTGTCATGTCCCAGCCTATGGGAGTAGTGCCT192    ValGluAspHisGlyValValMetSerGlnProMetGlyValValPro    505560    GGTACTGCCAACACCTCAGTTGTGACC219    GlyThrAlaAsnThrSerValValThr    6570    (2) INFORMATION FOR SEQ ID NO: 31:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 223    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: Not Relevant    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:    LeuGlnLeuGluAlaGluAsnTyrGluGlyHisThrProLeuHisVal    151015    AlaValIleHisLysAspValGluMetValArgLeuLeuArgAspAla    202530    GlyAlaAspLeuAspLysProGluProThrCysGlyArgSerProPhe    354045    IleTrpLeuAspLeuGluAlaArgAsnTyrAspGlyLeuThrAlaLeu    505560    HisValAlaValAsnThrGluCysGlnGluThrValGlnLeuLeuLeu    65707580    GluArgGlyAlaAspIleAspValAspIleLysSerGlyArgSerPro    859095    LeuIleHisGlnTrpArgProGlySerArgCysAlaGlyAlaSerLeu    100105110    ArgAlaGlyAlaAsnProAlaAlaArgMetTyrGlyGlyArgThrPro    115120125    LeuGlySerAlaMetLeuArgProAsnProIleLeuAlaArgLeuLeu    130135140    ArgAlaValGluAsnAsnSerLeuSerMetValGlnLeuLeuLeuGln    145150155160    HisGlyAlaAsnValAsnAlaGlnMetSerGlySerSerAlaLeuHis    165170175    SerAlaSerGlyArgGlyLeuLeuProLeuValArgThrLeuValAla    180185190    HisGlyAlaProGluProGluGlyLysAspGluLysSerGlyProArg    195200205    SerGlyAlaAspSerSerLeuLysAsnCysHisAsnAspThrPro    210215220    __________________________________________________________________________

What is claimed is:
 1. A substantially pure polypeptide comprising anamino acid sequence that is at least 80% identical to the amino acidsequence of JL1 shown in FIG. 2 (SEQ ID NO: 1) and which specificallyand physically interacts with a thyroid hormone receptor in an in vivointeraction trap assay.
 2. The polypeptide of claim 1, wherein saidpolypeptide is derived from a mammal.
 3. The polypeptide of claim 2,wherein said mammal is a human.
 4. The substantially pure polypeptide ofclaim 1, said polypeptide being produced by expression of a purified DNAencoding said polypeptide.
 5. A therapeutic composition comprising as anactive ingredient a polypeptide according to claim 1, said activeingredient being formulated in a physiologically-acceptable carrier. 6.The substantially pure polypeptide of claim 1, said polypeptidecomprising the amino acid sequence of JL1 shown in FIG. 2 (SEQ ID NO:1).